Rice restorer gene to the rice BT type cytoplasmic male sterility

ABSTRACT

The purpose of the present invention is to provide the rice restorer gene to the rice BT type cytoplasmic male sterility. The gene of the present invention comprises a nucleic acid encoding the amino acid sequence of SEQ ID NO. 75, or an amino acid sequence which is identical to at least 70% of the amino acid sequence of SEQ ID NO. 75, and which functions to restore fertility. Preferably, the gene of the present invention has the base sequence of SEQ ID NOS:69-74, 80-85 or the bases 43907-46279 of SEQ ID NO:27.

FIELD OF THE INVENTION

The present invention relates to the rice restorer gene to the rice BTtype cytoplasmic male sterility.

The present application claims priority based on Japanese PatentApplication No. 2002-107560 filed on Jul. 5, 2002. The entiredisclosures of the patent application are incorporated herein.

PRIOR ART

Rice is a self-fertilizing plant, so in order to perform crossingbetween varieties, self-fertilization must first be avoided by removingall stamens in a glumaceous flower just before flowering and, thenfertilization is effected with pollens from the parent variety withwhich it is to be crossed. However, this manual crossing method isentirely unsuitable for producing a large quantity of hybrid seeds on acommercial scale.

Accordingly, hybrid rice is produced by the triple-crossing system whichmakes use of cytoplasmic male sterility. In the triple-crossing system,the following three lines are employed, i.e., a sterile line having malesterile cytoplasm, a restorer line having Rf-1 gene and a maintainerline having the same nuclear gene as that of the sterile line but nothaving any sterile cytoplasm. By using these three lines, (i) hybridseeds can be obtained through fertilization of the sterile line with thepollen of the restorer line whereas (ii) the sterile line can bemaintained through its fertilization with the pollen of the maintainerline.

When employing the BT type male sterile cytoplasm in the triple-crossingsystem, it is important to breed rice of the restorer line and to thisend, it is necessary to ensure that the rice at every stage of breedingmaintains Rf-1 gene and that the Rf-1 gene is homozygous at the finalstage. It also becomes necessary in the triple-crossing system to checkto ensure that the variety used as the restorer line possesses Rf-1gene, or to check for the presence of Rf-1 gene in order to ensure thatthe resulting hybrid seeds have restored fertility.

In order to genotype the locus of Rf-1 gene in a plant, it has beennecessary that F1 plants be first formed from hybrid seeds obtained bycrossing the plant to be genotyped to a standard line and thenself-fertilized, followed by investigating the incidence of individualsthat can produce seeds at a frequency higher than a certain level (e.g.70˜80% or more). The standard line refers to the maintainer line, thesterile line or a set of the two lines, and it is appropriately chosendepending upon whether the cytoplasm of the individual under test is ofBT type or normal type or unknown. If the standard line is a sterileline, it is crossed to the individual under test as the female parentand if the standard line is a maintainer line, it is crossed as the maleparent.

However, these techniques require a huge amount of labor and time tocarry out. As a further problem, fertilization for seed production issensitive to environmental factors and if an investigation is made in anunfavorable environment such as cold climate or insufficient daylight,sterility may be caused irrespective of the genotype constitution, withthe result that genotyping of the locus of Rf-1 gene cannot be performedaccurately.

With a view to solving these problems, it has recently been proposedthat Rf-1 gene be checked for its presence by a technique of molecularbiology. The technical idea of this technique lies in checking for thepresence or absence of Rf-1 gene by detecting base sequences linked toRf-1 gene (such sequences are hereunder referred to as DNA markers).Note that it is not possible to directly detect Rf-1 gene since the DNAsequence of Rf-1 gene has not been clarified so far.

For example, it has been reported that the locus of Rf-1 gene in rice ispresent on chromosome 10 and located between DNA marker (RFLP marker)loci G291 and G127 which can be used in restriction fragment lengthpolymorphism analysis (RFLP) (Fukuta et al., 1992, Jpn J. Breed. 42(supl. 1) 164-165). This is a known method of genotyping the locus ofRf-1 gene by investigating the genotypes of DNA marker loci G291 andG127 which are linked to Rf-1 gene.

However, the conventional molecular biology techniques have severalproblems. First, they use RFLP markers which need to be detected bySouthern blot analysis. In order to perform Southern blot analysis, DNAat the microgram level needs to be purified from the individual undertest and, in addition, there is a need to carry out a sequence of stepscomprising treatment with restriction enzymes, electrophoresis,blotting, hybridization with a probe and signal detection; this not onlyinvolves considerable labor but it also takes about one week to obtainthe test results.

The second problem is that since the gene map distance between RFLPmarker loci G291 and G127 is as long as about 30 cM (corresponding toabout 9000 kbp in rice DNA), the probability for the occurrence ofdouble recombination in the region would be a few percent and hence, itis not always guaranteed that the genotype of the locus of Rf-1 gene canbe estimated correctly by the markers.

Thirdly, when the presence of Rf-1 gene is estimated by detecting RFLPmarker loci G291 and G127, not only Rf-1 gene but also the gene regionbetween those loci are introduced into the fertility restorer lineselected as the result of breeding. As a consequence, the introduced DNAsequence will have a chromosomal region of 30 cM or longer from the Rf-1gene donor parent, and this presents the risk of introducing adeleterious gene that may potentially be present within that region.

In order to solve these problems, there have been developed a dominantDNA marker (Japanese Patent Public Disclosure No. 222588/1995) and aco-dominant DNA marker (Japanese Patent Public Disclosure No.313187/1997), both of which are linked to the locus of Rf-1 gene. Thesemarkers are linked to the locus of Rf-1 gene, their genetic distancesfrom Rf-1 gene respectively being 1.6±0.7 cM (corresponding to about 480kbp in rice DNA) and 3.7±1.1 cM (corresponding to about 1110 kbp in riceDNA), and their loci being on opposite sides of the locus of Rf-1 gene.Hence, the presence of Rf-1 gene can be estimated by detecting thepresence of both the locus of the dominant PCR marker and that of theco-dominant PCR marker. The use of the co-dominant PCR marker alsoenables us to estimate as to whether the locus of Rf-1 gene ishomozygous or heterozygous.

However, the use of these PCR markers still involve several problems.The co-dominant marker has a genetic distance of 3.7±1.1 cM from thelocus of Rf-1 gene, and the problem of potentially high frequency ofrecombination with the locus of Rf-1 gene has not been fully dissolved.As a result, speaking of the co-dominant marker itself, correctdetection can be made as to whether it is homozygous or a heterozygous.However, if recombination occurs between the locus of the co-dominantmarker and that of Rf-1 gene, the genotype of Rf-1 gene locus cannot bedetermined correctly, particularly as to whether it is homozygous orheterozygous. On the other hand, if the dominant marker is used togenotype the locus of Rf-1 gene, the marker will detect individualsindiscriminately irrespective of whether they are homozygous (Rf-1/Rf-1)or heterozygous (Rf-1/rf-1) with respect to Rf-1 gene. Therefore, evenif the co-dominant marker is used in combination with the dominantmarker in order to genotype the locus of Rf-1 gene, it is not possibleto correctly distinguish individuals having Rf-1 gene homozygously fromthose having the gene heterozygously. Further, if no amplificationproduct is obtained in PCR using the dominant marker, one cannot denythe possibility that this is due to some problems in the experimentalprocedure. As a further problem, since the genetic distance between theco-dominant marker and the dominant marker is as great as about 5.3 cM(around 1590 kbp), the size of the chromosomal region introduced fromthe Rf-1 gene donor parent cannot be limited to a sufficiently smallvalue to prevent any concomitant introduction of a deleterious genewhich may be contained in that region.

Japanese Patent Public Disclosure No. 139465/2000 describes co-dominantPCR markers that were developed on the basis of the base sequences ofRFLP markers located in the neighborhood of Rf-1 gene on chromosome 10of rice. However, most of those PCR markers are spaced from the Rf-1gene by a genetic distance greater than about 1 cM.

DISCLOSURE OF THE INVENTION

An object of the present invention is to provide methods for restoringrice fertility. A method of the present invention comprises introducinga nucleic acid into rice, wherein the nucleic acid encodes the aminoacid sequence of SEQ ID NO. 75, or an amino acid sequence which isidentical to at least 70% of the amino acid sequence of SEQ ID NO. 75,and which functions to restore fertility. In one of the preferredembodiments of the present invention, the nucleic acid encoding theamino acid sequence of SEQ ID NO. 75, or an amino acid sequence which isidentical to at least 70% of the amino acid sequence of SEQ ID NO. 75 isselected from nucleic acids of the following a)-p):

a) a nucleic acid comprising the bases 215-2587 of SEQ ID NO:69;

b) a nucleic acid comprising the bases 213-2585 of SEQ ID NO:70;

c) a nucleic acid comprising the bases 218-2590 of SEQ ID NO:71;

d) a nucleic acid comprising the bases 208-2580 of SEQ ID NO:72;

e) a nucleic acid comprising the bases 149-2521 of SEQ ID NO:73;

f) a nucleic acid comprising the bases 225-2597 of SEQ ID NO:74;

g) a nucleic acid comprising the bases 43907-46279 of SEQ ID NO:27;

h) a nucleic acid comprising the bases 229-2601 of SEQ ID NO:80;

i) a nucleic acid comprising the bases 175-2547 of SEQ ID NO:81;

j) a nucleic acid comprising the bases 227-2599 of SEQ ID NO:82;

k) a nucleic acid comprising the bases 220-2592 of SEQ ID NO:83;

l) a nucleic acid comprising the bases 174-2546 of SEQ ID NO:84;

m) a nucleic acid comprising the bases 90-2462 of SEQ ID NO:85;

n) a nucleic acid which is identical to at least 70% of the nucleic acidof any of a)-m), and which functions to restore fertility;

o) a nucleic acid which hybridizes to the nucleic acid of any of a)-m)under a moderate or high stringent condition, and which functions torestore fertility; and

p) a nucleic acid wherein one or a plurality of base(s) is deleted from,added to or substituted from the nucleic acid of any of a)-m), and whichfunctions to restore fertility.

Preferably, in the method of the present invention, the nucleic acidencoding the amino acid sequence of SEQ ID NO. 75, or an amino acidsequence which is identical to at least 70% of the amino acid sequenceof SEQ ID NO. 75, and which functions to restore fertility, meets atleast one of the following requirements 1)-12):

1) a base corresponding to the base 1769 of SEQ ID NO. 69 is A;

2) a base corresponding to the base 1767 of SEQ ID NO. 70 is A;

3) a base corresponding to the base 1772 of SEQ ID NO. 71 is A;

4) a base corresponding to the base 1762 of SEQ ID NO. 72 is A;

5) a base corresponding to the base 1703 of SEQ ID NO. 73 is A;

6) a base corresponding to the base 1779 of SEQ ID NO. 74 is A;

7) a base corresponding to the base 1783 of SEQ ID NO. 80 is A;

8) a base corresponding to the base 1729 of SEQ ID NO. 81 is A;

9) a base corresponding to the base 1781 of SEQ ID NO. 82 is A;

10) a base corresponding to the base 1774 of SEQ ID NO. 83 is A;

11) a base corresponding to the base 1728 of SEQ ID NO. 84 is A; or 12)a base corresponding to the base 1644 of SEQ ID NO. 85 is A.

Another object of the present invention is to provide a method fordiscerning whether a subject rice individual or a seed thereof has theRf-1 gene or not, utilizing a nucleic acid encoding the amino acidsequence of SEQ ID NO. 75, or an amino acid sequence which is identicalto at least 70% of the amino acid sequence of SEQ ID NO. 75, and whichfunctions to restore fertility. Preferably, in an embodiment of thepresent method, the subject rice individual or the seed thereof isdetermined to have the Rf-1 gene, in the case that the nucleic acidencoding the amino acid sequence of SEQ ID NO. 75, or an amino acidsequence which is identical to at least 70% of the amino acid sequenceof SEQ ID NO. 75, and which functions to restore fertility, meets atleast one of the following requirements 1)-12):

1) a base corresponding to the base 1769 of SEQ ID NO. 69 is A;

2) a base corresponding to the base 1767 of SEQ ID NO. 70 is A;

3) a base corresponding to the base 1772 of SEQ ID NO. 71 is A;

4) a base corresponding to the base 1762 of SEQ ID NO. 72 is A;

5) a base corresponding to the base 1703 of SEQ ID NO. 73 is A;

6) a base corresponding to the base 1779 of SEQ ID NO. 74 is A;

7) a base corresponding to the base 1783 of SEQ ID NO. 80 is A;

8) a base corresponding to the base 1729 of SEQ ID NO. 81 is A;

9) a base corresponding to the base 1781 of SEQ ID NO. 82 is A;

10) a base corresponding to the base 1774 of SEQ ID NO. 83 is A;

11) a base corresponding to the base 1728 of SEQ ID NO. 84 is A; or 12)a base corresponding to the base 1644 of SEQ ID NO. 85 is A.

Another object of the present invention is to provide a method forinhibiting the function of the Rf-1 gene to restore fertility. Theinhibition method of the present invention comprises, in an embodiment,introducing an antisense having at least 100 bases in length, and beingselected from base sequences complementary to a nucleic acid encodingthe amino acid sequence of SEQ ID NO. 75, or an amino acid sequencewhich is identical to at least 70% of the amino acid sequence of SEQ IDNO. 75, and which functions to restore fertility.

Still another object of the present invention is to provide a nucleicacid encoding the amino acid sequence of SEQ ID NO. 75, or an amino acidsequence which is identical to at least 70% of the amino acid sequenceof SEQ ID NO. 75, and which functions to restore fertility. The presentinvention provides, in an embodiment, a nucleic acid selected fromnucleic acids of the following a)-p):

a) a nucleic acid comprising the bases 215-2587 of SEQ ID NO:69;

b) a nucleic acid comprising the bases 213-2585 of SEQ ID NO:70;

c) a nucleic acid comprising the bases 218-2590 of SEQ ID NO:71;

d) a nucleic acid comprising the bases 208-2580 of SEQ ID NO:72;

e) a nucleic acid comprising the bases 149-2521 of SEQ ID NO:73;

f) a nucleic acid comprising the bases 225-2597 of SEQ ID NO:74;

g) a nucleic acid comprising the bases 43907-46279 of SEQ ID NO:27;

h) a nucleic acid comprising the bases 229-2601 of SEQ ID NO:80;

i) a nucleic acid comprising the bases 175-2547 of SEQ ID NO:81;

j) a nucleic acid comprising the bases 227-2599 of SEQ ID NO:82;

k) a nucleic acid comprising the bases 220-2592 of SEQ ID NO:83;

l) a nucleic acid comprising the bases 174-2546 of SEQ ID NO:84;

m) a nucleic acid comprising the bases 90-2462 of SEQ ID NO:85;

n) a nucleic acid which is identical to at least 70% of the nucleic acidof any of a)-m), and which functions to restore fertility;

o) a nucleic acid which hybridizes to the nucleic acid of any of a)-m)under a moderate or high stringent condition, and which functions torestore fertility; and

p) a nucleic acid wherein one or a plurality of base(s) is deleted from,added to or substituted from the nucleic acid of any of a)-m), and whichfunctions to restore fertility.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1 shows the results of chromosomal walking started from the RFLPmarker locus S12564.

FIG. 2 shows an alignment of lambda clone contigs in relation to the BACclone AC068923.

FIG. 3 shows the chromosomal organization of recombinant pollensproximal to the Rf-1 locus (all fertile) as mapped in close proximity tothe Rf-1 locus based on the genotypes at the marker loci of 10individuals (RS1, RS2, RC1-8) generated from the pollens. White barsrepresent japonica regions and black bars represent indica regions.

FIG. 4 is a gene map in which the locus of Rf-1 gene on chromosome 10 ofrice is positioned on a linkage map in relation to various markers; thevalues of map distance were calculated from the segregation data from1042 F1 individuals.

FIG. 5 shows fragments from 10 genomic clones used for theidentification of the Rf-1 region by complementation assays. Lambdaclones obtained by chromosomal walking (thin lines) were used forcomplementation assays of the chromosomal regions shown by bold lines.XSF18 was found to contain a deletion shown by dotted line.

FIG. 6 shows the results of complementation assays using a 15.7 kbfragment from XSG16 (Example 10) and a 16.2 kb fragment from XSF18(Example 8). The plant transformed with the 15.7 kb fragment from XSG16has restored fertility as proved by ears bowing.

FIG. 7 is a schematic picture showing the Rf-1 gene structure. Whitebars and black bars represent exons and introns, respectively. Numbersof base pairs are shown for the exon portions.

FIG. 8 is a schematic picture showing positional relationships betweenthe IR24 genome fragment subjected to the complementation assays, probesused for the cDNA library screening and the Rf-1 gene deduced from theisolated cDNAs. White bars and black bars in the Rf-1 gene representexons and introns, respectively. Numbers of base pairs are shown for theexon portions.

BEST MODES FOR PERFORMING THE INVENTION

We began by restricting the Rf-1 locus to a very small region onchromosome 10. On this basis, we developed PCR markers proximal to theRf-1 locus and found a method for detecting the Rf-1 gene by utilizingon the linkage of these PCR markers to the Rf-1 locus. Specifically, thepresence of the Rf-1 gene is tested and individuals homozygous for theRf-1 gene are selected by genotyping at the novel PCR marker lociproximal to the Rf-1 locus on the basis that the Rf-1 locus is mappedbetween the PCR marker loci S12564 Tsp509I and C1361 MwoI on chromosome10 of rice. We previously filed a patent application for the method fordetecting the Rf-1 gene under Japanese Patent Application No.2000-247204 on Aug. 17, 2000. The entire disclosure of the patentapplication is incorporated herein by reference.

I. Methods for Estimating the Genotype at the Rf-1 Locus Described inJapanese Patent Application No. 2000-247204

Japanese Patent Application No. 2000-247204 describes methods fordetermining whether or not a rice individual or seed under test has theRf-1 gene on the basis that the Rf-1 locus is mapped between the PCRmarker loci S12564 and C1361 on chromosome 10 of rice.

Markers

Primer pairs designed to be specific to particular regions near thelocus of Rf-1 gene are used in PCR and the amplification products aretreated with particular restriction enzymes; upon electrophoresis, riceof indica lines in some cases provide an observable band of a differentsize from that of rice of Japonica lines. This band which ischaracteristic of indica lines is herein referred to as the Rf-1 linkedband. Now that it has been made clear by the present inventors that thelocus of Rf-1 gene is located between PCR markers S12564 Tsp509I andC1361 MwoI on chromosome 10 of rice, the skilled artisan canappropriately develop and employ PCR markers that are present in theneighborhood of Rf-1 gene.

For instance, according to the invention, a rice individual under testis checked to see if its genome contains at least one of the PCR markerslisted below, thereby determining whether the individual under test hasRf-1 gene linked to those PCR markers:

(1) marker 1: PCR marker R1877 EcoRI which, when rice genomic DNA issubjected to PCR with DNA primers having the sequences of SEQ ID NO:1and SEQ ID NO:2, can detect polymorphisms between rice individuals ofthe japonica and indica lines depending on whether the amplificationproducts have a recognition site for restriction enzyme EcoRI;

(2) marker 2: PCR marker G4003 HindIII (SEQ ID NO:19) which, when ricegenomic DNA is subjected to PCR with DNA primers having the sequences ofSEQ ID NO:3 and SEQ ID NO:4, can detect polymorphisms between riceindividuals of the japonica and indica lines depending on whether theamplification products have a recognition site for restriction enzymeHindIII;

(3) marker 3: PCR marker C1361 MwoI (SEQ ID NO:20) which, when ricegenomic DNA is subjected to PCR employing DNA primers having thesequences of SEQ ID NO:5 and SEQ ID NO:6, can detect polymorphismsbetween rice individuals of the japonica and indica lines depending onwhether the amplification products have a recognition site forrestriction enzyme MwoI;

(4) marker 4: PCR marker G2155 MwoI (SEQ ID NO:21) which, when ricegenomic DNA is subjected to PCR with DNA primers having the sequences ofSEQ ID NO:7 and SEQ ID NO:8, can detect polymorphisms between riceindividuals of the japonica and indica lines depending on whether theamplification products have a recognition site for restriction enzymeMwoI;

(5) marker 5: PCR marker G291 MspI (SEQ ID NO:22) which, when ricegenomic DNA is subjected to PCR with DNA primers having the sequences ofSEQ ID NO:9 and SEQ ID NO:10, can detect polymorphisms between riceindividuals of the japonica and indica lines depending on whether theamplification products have a recognition site for restriction enzymeMspI;

(6) marker 6: PCR marker R2303 BslI (SEQ ID NO:23) which, when ricegenomic DNA is subjected to PCR with DNA primers having the sequences ofSEQ ID NO:11 and SEQ ID NO:12, can detect polymorphisms between riceindividuals of the japonica and indica lines depending on whether theamplification products have a recognition site for restriction enzymeBslI;

(7) marker 7: PCR marker S10019 BstUI (SEQ ID NO:24) which, when ricegenomic DNA is subjected to PCR with DNA primers having the sequences ofSEQ ID NO:13 and SEQ ID NO:14, can detect polymorphisms between riceindividuals of the japonica and indica lines depending on whether theamplification products have a recognition site for restriction enzymeBstUI;

(8) marker 8: PCR marker S10602 KpnI (SEQ ID NO:25) which, when ricegenomic DNA is subjected to PCR with DNA primers having the sequences ofSEQ ID NO:15 and SEQ ID NO:16, can detect polymorphisms between riceindividuals of the japonica and indica lines depending on whether theamplification products have a recognition site for restriction enzymeKpnI; and

(9) marker 9: PCR marker S12564 Tsp509I (SEQ ID NO:26) which, when ricegenomic DNA is subjected to PCR with DNA primers having the sequences ofSEQ ID NO:17 and SEQ ID NO:18, can detect polymorphisms between riceindividuals of the japonica and indica lines depending on whether theamplification products have a recognition site for restriction enzymeTsp509I.

Assuming that the locus of Rf-1 gene was highly likely to be locatednear the nine RFLP marker regions R1877, G291, R2303, S12564, C1361,S10019, G4003, S10602 and G2155 on chromosome 10 of rice (see theresults of RFLP linkage analysis described in Fukuta et al., 1992, Jpn.J. Breed. 42 (supl. 1) 164-165 and the RFLP linkage map of ricedescribed in Harushima et al., 1998, Genetics, 148, 479-494), thepresent inventors converted those RFLP markers to co-dominant PCRmarkers such as CAPS markers or dCAPS markers as described below inReference example 1 (Michaels and Amasino, 1998, The Plant Journal,14(3), 381-385; Neff et al., 1998, The Plant Journal, 14(3), 387-392).As a result of this conversion, the PCR markers above have beenobtained.

Among these PCR markers, one group consisting of PCR markers R1877EcoRI, G291 MspI (SEQ ID NO:22), R2303 BslI (SEQ ID NO:23) and S12564Tsp509I (SEQ ID NO:26) and the other group consisting of PCR markersC1361 MwoI (SEQ ID NO:20), S10019 BstUI (SEQ ID NO:24), G4003 HindIII(SEQ ID NO:19), S10602 KpnI (SEQ ID NO:25) and G2155 MwoI (SEQ ID NO:21)are on opposite sides of the locus of Rf-1 gene on chromosome 10 ofrice.

Therefore, in one embodiment, the presence of the Rf-1 gene is detectedby detecting Rf-1 linked bands by (a) at least one PCR marker selectedfrom the group consisting of PCR markers R1877 EcoRI, G291 MspI, R2303BslI and S12564 Tsp509I, and (b) at least one PCR marker selected fromthe group consisting of PCR markers C1361 MwoI, S10019 BstUI, G4003HindIII, S10602 KpnI and G2155 MwoI. In this case, at least S12564Tsp509I from group (a) and at least C1361 MwoI from group (b) arepreferably used as the closest PCR markers to the Rf-1 gene. If Rf-1linked bands are detected with PCR markers of both (a) and (b) in thegenome of the rice under test, it can be estimated with a highprobability that the rice contains Rf-1 gene.

In another embodiment, Rf-1 linked bands are detected by at least twoPCR markers of group (a) and at least two PCR markers of group (b)above. For example, a rice individual carrying the Rf-1 gene with aminimum of unwanted gene regions can be selected by picking up anindividual in which Rf-1 linked bands are detected by markers of groups(a) and (b) more proximal to the Rf-1 gene but not detected by markersof groups (a) and (b) more distal from the Rf-1 gene on the gene mapshown in FIG. 1. Again, it is preferred that at least one PCR marker ofgroup (a) is S12564 Tsp509I and at least one PCR marker of group (b) isC1361 MwoI. Thus, the two PCR marker loci S12564 Tsp509I and C1361 MwoIare separated by a genetic distance of 0.3 cM. By utilizing thischaracteristic, the chromosomal region that is introduced from the Rf-1gene donor parent can be narrowed down to a size of about 1 cM. Thishelps minimize the possibility of introducing into the restorer line adeleterious gene that may be present in the neighborhood of Rf-1 gene inthe donor parent.

Detection of the Rf-1 Gene

In order to detect Rf-1 gene in the genome of a rice under test, any oneof the above PCR markers is amplified from the genome of the rice by PCRusing primers of SEQ ID NOS: 1-18 above and then detected by thepolymerase chain reaction-restriction fragment length polymorphismmethod (PCR-RFLP). PCR-RFLP is a method that is applicable to the casewhere polymorphisms exist among variety lines at recognition sites ofrestriction enzymes in the sequences of PCR amplified DNA fragments andby which specific polymorphisms can conveniently be identified on thebasis of cleavage patterns with those restriction enzymes (D. E. Harryet al., Theor. Appl. Genet. (1998), 97:327-336)

Restriction enzyme cleavage patterns show the bands as shown in Table 1below on a visualized gel depending on the primer pair used.

TABLE 1 Approximate size (bp) of detected band Detection of marker 1(R1877 EcoRI) with primer pair 1 When the genome of test rice has 1500and 1700 Rf-1 gene homozygously: When the genome of test rice has 1500,1700 and Rf-1 gene heterozygously: 3200 When the genome of test rice has3200 no Rf-1 gene: Detection of marker 2 (G4003 HindIII) with primerpair 2 When the genome of test rice has 362 Rf-1 gene homozygously: Whenthe genome of test rice has 95, 267 and 362 Rf-1 gene heterozygously:When the genome of test rice has 95 and 267 no Rf-1 gene: Detection ofmarker 3 (C1361 MwoI) with primer pair 3 When the genome of test ricehas 50 and 107 Rf-1 gene homozygously: When the genome of test rice has25, 50, 79 and 107 Rf-1 gene heterozygously: When the genome of testrice has 25, 50 and 79 no Rf-1 gene: Detection of marker 4 (G2155 MwoI)with primer pair 4 When the genome of test rice has 25, 27 and 78 Rf-1gene homozygously: When the genome of test rice has 25, 27, 78 and 105Rf-1 gene heterozygously: When the genome of test rice has 25 and 105 noRf-1 gene: Detection of marker 5 (G291 MspI) with primer pair 5 When thegenome of test rice has 25, 49 and 55 Rf-1 gene homozygously: When thegenome of test rice has 25, 49, 55 and 104 Rf-1 gene heterozygously:When the genome of test rice has 25 and 104 no Rf-1 gene: Detection ofmarker 6 (R2303 BslI) with primer pair 6 When the genome of test ricehas 238, 655 and 679 Rf-1 gene homozygously: When the genome of testrice has 238, 655, 679 and Rf-1 gene heterozygously: 1334 When thegenome of test rice has 238 and 1334 no Rf-1 gene: Detection of marker 7(S10019 BstUI) with primer pair 7 When the genome of test rice has 130,218 and 244 Rf-1 gene homozygously: When the genome of test rice has130, 218, 244 and Rf-1 gene heterozygously: 462 When the genome of testrice has 130 and 462 no Rf-1 gene: Detection of marker 8 (S10602 KpnI)with primer pair 8 When the genome of test rice has 724 Rf-1 genehomozygously: When the genome of test rice has 117, 607 and 724 Rf-1gene heterozygously: When the genome of test rice has 117 and 607 noRf-1 gene: Detection of marker 9 (S12564 Tsp509I) with primer pair 9When the genome of test rice has 41 and 117 Rf-1 gene homozygously: Whenthe genome of test rice has 26, 41, 91 and 117 Rf-1 gene heterozygously:When the genome of test rice has 26, 41 and 91 no Rf-1 gene:II. Identification of the Rf-1 Locus

As described above, Japanese Patent Application No. 2000-247204discloses RFLP-PCR markers based on our finding that the Rf-1 locus ismapped between DNA marker loci S12564 Tsp509I and C1361 MwoI.Fertility-restoring lines are established by backcrossing the Rf-1 geneinto a normal japonica variety not containing the Rf-1 gene. If themethod for identifying the Rf-1 locus described in Japanese PatentApplication No. 2000-247204 is used during this process, not only therestoring lines can be established efficiently (within 2-3 years) butalso the length of insert fragments can be controlled.

However, introduction by crossing inevitably introduce regions proximalto Rf-1 at the same time. Japanese Patent Application No. 2000-247204showed that the Rf-1 locus is mapped between DNA marker loci S12564Tsp509I and C1361 MwoI, but the distance between both loci is about 0.3cM, i.e. about 90 kbp. If a deleterious gene existed proximal to Rf-1,it would be undeniable that the deleterious gene might be insertedtogether with the Rf-1 gene.

Thus, we searched for regions linked to the Rf-1 gene between DNA markerloci S12564 Tsp509I and C1361 MwoI by chromosomal walking and geneticanalysis based on the close linkage between the Rf-1 locus and the DNAmarker locus S12564 Tsp509I. As a result, we successfully identified theregion of the Rf-1 locus including the Rf-1 gene up to about 76 kb anddetermined the entire base sequence of said region. According to thepresent invention, it is possible to introduce the function of afertility restorer gene into BT male sterile cytoplasms by geneticengineering techniques.

Specifically, in Japanese Patent Application No. 2000-247204, linkageanalyses on a population of 1042 individuals prepared by pollinating MSKoshihikari with MS-FR Koshihikari (heterozygous at the Rf-1 locus)revealed one recombinant between the Rf-1 and S12564 Tsp509I loci andtwo recombinants between the Rf-1 and C1361 MwoI loci (Referenceexamples 1-2 herein). In the present invention, 4103 individuals wereadded to the population to analyze a total of 5145 individuals. As aresult, one recombinant between the Rf-1 and S12564 Tsp509I loci and sixrecombinants between the Rf-1 and C1361 MwoI loci were newly found witha total of 2 and 8 recombinants. These 10 individuals were tested by thehigh-precision segregation analysis of the present invention asrecombinants proximal to the Rf-1 locus (Example 1).

The frequency of 8 recombinants between the Rf-1 and C1361 MwoI loci ascompared with 2 recombinants between the Rf-1 and S12564 Tsp509I locimeans that the S12564 Tsp509I locus is genetically closer to the Rf-1locus than the C1361 MwoI locus. Genetic distance (expressed inrecombination frequency: cM) and physical distance (expressed in thenumber of base pairs: bp) are not always proportional to each other, butit can be normally expected that physical distance decreases withgenetic distance.

Thus, we tried to isolate the Rf-1 locus by chromosomal walking startedfrom the S12564 Tsp509I locus (Example 2). Chromosomal walking wasperformed on a genomic library prepared from λ DASH II vector using thegenomic DNA of an indica variety IR24 and a japonica variety Asominori.IR24 is a variety carrying Rf-1, while Asominori is a variety notcarrying Rf-1. As a result of chromosomal walking, contigs covering achromosomal region of about 76 kb (ordered sets of overlapping clones ona chromosome) were able to be prepared from genomic clones of IR24, andthe entire base sequence (76363 bp) thereof was determined.

Then, 12 markers were newly developed on the basis of the base sequencedata or the like obtained and a high-precision segregation analysis wasperformed on the 10 recombinants proximal to Rf-1 locus described above(Example 3). As a result, a 65 kb sequence included in the chromosomalregion of about 76 kb above was shown to contain a sequence determiningthe presence of the function of the Rf-1 gene. This region is covered bya contig consisting of 8 genomic clones. Each clone has a length ofabout 12-22 kb and has overlapping domains of at least 4.7 kb. Genes forrice are known to have a wide range of lengths (from short ones to largeones), but most of them seem to have a length of several kb or less.Thus, at least one of these 8 genomic clones is expected to contain thefull-length Rf-1 gene.

We further restricted the Rf-1 gene region in the chromosomal region ofabout 76 kb above and performed complementation assays to directlydemonstrate the presence of a fertility restoring ability.

Specifically, 10 partial fragments (each 10-21 kb) in the above regionof 76 kb were separately introduced into immature seeds of a malesterility line MS Koshihikari by genetic engineering techniques (FIG.5). Of the 10 partial fragments used, 8 fragments are derived from 8genomic clones previously obtained by chromosomal walking (XSE1, XSE7,XSF4, XSF20, XSG22, XSG16, XSG8 and XSH18 shown in FIG. 1 and describedin Example 3). Additionally, fragments derived from 2 clones XSF18 andXSX1 were also analyzed by complementation assays. XSF18 is identical toXSF20 at the 5′ and 3′ ends (bases 20328 and 41921 of SEQ ID NO:27,respectively), but lacks internal bases 33947-38591. This is becauseclone XSF18 was initially isolated but found to contain the abovedeletion during amplification after isolation, and therefore, theamplification step was freshly taken to isolate a complete clonedesignated XSF20 (Example 8). XSX1 is a clone freshly prepared fromclones XSG8 and XSH18 by restriction enzyme treatment and ligation tocontain sufficient overlapping domains because of the overlappingdomains of both clones are relatively small (about 7 kb) (Example 13).

If the insert fragment completely contains the Rf-1 gene, transformedindividuals at this generation restore fertility because Rf-1 is adominant gene. In complementation assays plants transformed with eachfragment were evaluated for seed fertility to find that thosetransformed with a 15.6 kb fragment (including bases 38538-54123 of SEQID NO:27) derived from the λ phage clone XSG16 restored seed fertility(Example 10). Plants transformed with the other fragments were allsterile. These results showed that the above 15.6 kb fragment completelycontains the Rf-1 gene. Moreover, a method for introducing the Rf-1 geneby genetic engineering techniques was provided by the present inventionand demonstrated to be effective.

To further specify the region of the λ phage clone XSG16 in which theRf-1 gene is contained, we evaluated seed fertility of shorter fragmentsthan the 15.6 kb fragment (including bases 38538-54123 of SEQ ID NO:27)by complementation assays. As a result, plants transformed with a 11.4kb fragment derived from XSG16 (including bases 42357-53743 of SEQ IDNO:27) were shown to restore seed fertility (Example 10(2)). Plantstransformed with a further shorter 6.8 kb fragment (including bases42132-48883 of SEQ ID NO:27) also restored seed fertility (Example10(3)). These results showed that the above 6.8 kb fragment contains theRf-1 gene.

The present inventors further continued studying, and identified thenucleic acid having the function to restore fertility. The amino acidsequence encoded by the nucleic acid then has been clarified.Specifically, DNA fragments corresponding to bases 43733-44038 and48306-50226 of SEQ ID NO:27 were first prepared by using PCR asdescribed in Examples 14-15. The cDNA library prepared from the linewherein Rf-1 is introduced to Koshihikari was screened by using theabove two DNA fragments as probes (Probe P and Q). As a result, terminalbase sequences of 6 clones are identical to the sequence of XSG16, andthese 6 clones were isolated as those containing the Rf-1 gene, and basesequences thereof were analyzed (SEQ ID NOS:69-74).

All of the sequences, SEQ ID NOS:69-74 encode a protein having the aminoacids 1-791 of SEQ ID NO:75. Specifically, all and each of the 215-2587of SEQ ID NO:69, the bases 213-2585 of SEQ ID NO:70, the bases 218-2590of SEQ ID NO:71, the bases 208-2580 of SEQ ID NO:72, the bases 149-2521of SEQ ID NO:73 and the bases 225-2597 of SEQ ID NO:74 encodes a proteinhaving amino acids 1-791 of SEQ ID NO:75. The above base sequencescorrespond to the bases 43907-46279 of SEQ ID NO:27.

The amino acid sequence of SEQ ID NO:75 was compared with the presumedamino acid sequence of the corn fertility restorer gene (Rf2), and theN-terminal 7 amino acid residues (Met-Ala-Arg-Arg-Ala-Ala-Ser) in bothamino acid sequences were concurred. These 7 amino acid residues areconsidered to be a portion of a targeting signal to mitochondria (Liu etal., 2001). Based on the above facts, the cDNAs isolated on thisoccasion are considered to contain the full coding region of the Rf-1gene. No homology between the amino acid sequences of the rice Rf-1 andthe corn Rf-2 can be found except for the above region.

In addition, the sequences of cDNAs isolated on this occasion werecompared with the genome sequence of IR24 (SEQ ID NO:27), and thestructures of exons and introns of the Rf-1 gene were clarified (FIG.7). As a result, it was shown that various transcription productswherein the splicing patterns and the poly A addition positions aredifferent, are present in a plant body. There is no intron in the codingregion of the Rf-1 gene.

As for the 6.8 kb fragment which restored seed fertility in thecomplementary assay of Example 10 (3), the present inventors furtherpursued a complementary assay. Specifically, in Example 16, a 4.2 kbfragment (the bases 42132-46318 of SEQ ID NO:27) containing the promoterregion and the presumed translation region of the Rf-1 gene within theabove 6.8 kb fragment was subjected to a complementary assay, and the4.2 kb fragment restored the seed fertility.

Further, in Example 17, six new clones containing the nucleic acidhaving the fertility restorer function were obtained. Specifically, PCRwas performed by using two primers corresponding to the bases45522-45545 and 45955-45932 of SEQ ID NO:27, and the genomic clone XSG16of IR24 as a template to obtain a DNA fragment. Plaque hybridizationassays were performed by using the DNA fragment as Probe R and the abovementioned Probe P. Six clones were newly obtained (#7-#12) from plaqueswhich are positive for both Probe P and Probe R. The results were shownin SEQ ID NOS:80-85.

All of the sequences, SEQ ID NOS:80-85 are presumed to encode a proteinhaving the amino acids 1-791 of SEQ ID NO:75. Specifically, all and eachof the 229-2601 of SEQ ID NO:80, the bases 175-2547 of SEQ ID NO:81, thebases 227-2599 of SEQ ID NO:82, the bases 220-2592 of SEQ ID NO:83, thebases 174-2546 of SEQ ID NO:84 and the bases 90-2462 of SEQ ID NO:85encodes a protein having amino acids 1-791 of SEQ ID NO:75. The abovebase sequences correspond to the bases 43907-46279 of SEQ ID NO:27.

The sequences of cDNAs isolated on this occasion were compared with thegenome sequence of IR24 (The Japanese Patent Application No.2001-285247, SEQ ID NO:27), and the structures of exons and introns wereclarified (FIG. 8). Among the cDNAs isolated on this occasion, there arethree cDNAs which do not have any exons irrelevant to the presumedtranslation region, and consist of a single exon (#10-#12, SEA ID NOS:83-85).

III. Nucleic Acids Containing the Rf-1 Locus

The present invention provides nucleic acids containing the locus of afertility restorer gene (Rf-1). The nucleic acids containing the locusof a fertility restorer gene (Rf-1) of the present invention include anucleic acid having the base sequence of SEQ ID NO. 27, or a nucleicacid having a base sequence which is identical to at least 70% of thebase sequence of SEQ ID NO. 27, and which functions to restorefertility. Further, as described in Example 10, it was confirmed thatthe Rf-1 gene is completely contained in especially the bases38538-54123 of the base sequence of SEQ ID NO:27. Still further, theregion containing the Rf-1 gene is determined to be, preferably thebases 38538-54123 of SEQ ID NO:27, more preferably the bases42357-53743, still preferably the bases 42132-48883, and still morepreferably the bases 42132-46318.

The present inventors further pursued the study, and determined that thefollowing regions as being nucleic acids containing the Rf-1 gene.

a) the bases 215-2587 of SEQ ID NO:69;

b) the bases 213-2585 of SEQ ID NO:70;

c) the bases 218-2590 of SEQ ID NO:71;

d) the bases 208-2580 of SEQ ID NO:72;

e) the bases 149-2521 of SEQ ID NO:73;

f) the bases 225-2597 of SEQ ID NO:74;

h) the bases 229-2601 of SEQ ID NO:80;

i) the bases 175-2547 of SEQ ID NO:81;

j) the bases 227-2599 of SEQ ID NO:82;

k) the bases 220-2592 of SEQ ID NO:83;

l) the bases 174-2546 of SEQ ID NO:84; and

m) the bases 90-2462 of SEQ ID NO:85.

The above base sequences correspond to g) the bases 43907-46279 of SEQID NO:27, and all of the bases encode the amino acid sequence 1-791 ofSEQ ID NO:75.

Hereinafter, in the present specification, the term “the base sequenceof SEQ ID NO:27” refers to the whole SEQ ID NO:27 or a portion thereofwhich takes part in the fertility restorer function, especially thebases 38538-54123. The term refers to more preferably the bases42357-53743, still preferably the bases 42132-48883, and still morepreferably the bases 42132-46318. And most preferably, it refers to g)the bases 43907-46279 of SEQ ID NO:27, or alternatively, a) the bases215-2587 of SEQ ID NO:69, b) the bases 213-2585 of SEQ ID NO:70, c) thebases 218-2590 of SEQ ID NO:71, d) the bases 208-2580 of SEQ ID NO:72,e) the bases 149-2521 of SEQ ID NO:73, f) the bases 225-2597 of SEQ IDNO:74, h) the bases 229-2601 of SEQ ID NO:80, i) the bases 175-2547 ofSEQ ID NO:81, j) the bases 227-2599 of SEQ ID NO:82, k) the bases220-2592 of SEQ ID NO:83, l) the bases 174-2546 of SEQ ID NO:84 or m)the bases 90-2462 of SEQ ID NO:85 corresponding thereto.

In the examples below, a nucleic acid was isolated from a genomiclibrary of indica rice IR24 containing the Rf-1 gene as a nucleic acidcontaining a fertility restorer gene (Rf-1) and determined to have thebase sequence of SEQ ID NO:27. However, the nucleic acid containing afertility restorer gene (Rf-1) of the present invention can be derivedfrom any indica variety carrying the Rf-1 gene. The indica varietiescarrying the Rf-1 gene include, but not specifically limited to, e.g.IR24, IR8, IR36, IR64, Chinsurah and BoroII. Known japonica varietiesnot carrying the Rf-1 gene include, but not limited to, Asominori,Koshihikari, Kirara 397, Akihikari, Akitakomachi, Sasanishiki,Kinuhikari, Nipponbare, Hatsuboshi, Koganebare, Hinohikari, Mineasahi,Aichinokaori, Hatsushimo, Akebono, Fujihikari, Minenoyukimochi,Kokonoemochi, Fukuhibiki, Dontokoi, Gohyakumangoku, Hanaechizen,Todorokiwase, Haenuki, Domannaka, Yamakikari, etc. The “indica” and“japonica” varieties are well known to those skilled in the art and therice varieties encompassed by the present invention can be readilydetermined by those skilled in the art.

Nucleic acids of the present invention include DNA in bothsingle-stranded and double-stranded forms, as well as the RNA complementthereof. DNA includes, for example, genomic DNA (including correspondingcDNA), chemically synthesized DNA, DNA amplified by PCR, andcombinations thereof.

Nucleic acids containing the Rf-1 gene of the present inventionpreferably have the base sequence of SEQ ID NO:27. More than one codonmay encode the same amino acid, and this is called degeneracy of thegenetic code. Thus, a DNA sequence not completely identical to SEQ IDNO:27 may encode a protein having an amino acid sequence completelyidentical to SEQ ID NO:27. Such a variant DNA sequence may result fromsilent mutation (e.g., occurring during PCR amplification), or can be aproduct of deliberate mutagenesis of a native sequence.

Preferably, the Rf-1 gene of the present invention encodes the aminoacid sequence described in SEQ ID NO:75. However, it is not limitedthereto, and may encode an amino acid sequence wherein one or more aminoacid residues are deleted, added or substituted.

The protein of the present invention is intended to include anyhomologous proteins as long as they have the fertility restorerfunction. The “amino acid variation” occurs at one or a plurality ofamino acids residues, preferably 1-20, more preferably 1-10, mostpreferably 1-5 amino acid residues. The amino acid sequence encoded bythe Rf-1 gene has an identity of at least about 70%, preferably about80% or more, more preferably about 90% or more, still preferably about95% or more, and most preferably about 98% or more with the amino acidsequence of SEQ ID NO:75.

The percent identity of the amino acids can be determined by visualinspection and mathematical calculation. The percent identity betweentwo protein sequences may be determined by comparing sequenceinformation based on the algorithm of Needleman, S. B. and Wunsch, C. D.(J. Mol. Biol., 48: 443-453, 1970) and using the GAP computer programavailable from University of Wisconsin Genetics Computer Group (UWGCG).The preferred default parameters for the “GAP” program include: (1) ascoring matrix as described in Henikoff, S and Henikoff, J. G. (Proc.Natl. Acad. Sci. USA, 89: 10915-10919, 1992), blosum 62; (2) a penaltyof 12 for each gap; (3) a penalty of 4 for each length of each gap; and(4) no penalty for end gaps.

Other programs used by those skilled in the art for sequence comparisoncan also be used. For example, the percent identity may be determined bycomparing sequence information using the BLAST program described inAltschul et al. (Nucl. Acids. Res. 25., p. 3389-3402, 1997). The programis available from the web site of National Center for BiotechnologyInformation (NCBI), or the web site of DNA Data bank of Japan (DDBJ) onthe Internet. Various factors (parameters) for the homology research viathe BLAST program are described in detail on the sites. A research isgenerally performed by using the default parameters, although somesetting may be appropriately modified.

It is well known for those skilled in the art that even proteins havingthe same function may have different amino acid sequences depending onthe varieties from which they are derived. The Rf-1 gene of the presentinvention includes such homologs and variants of the base sequence ofSEQ ID NO:27 so far as they function to restore fertility. Theexpression “function to restore fertility” means that fertility isconferred on a rice individual or seed when such a DNA fragment isintroduced. Fertility restoration may result from the expression of aprotein by the Rf-1 gene or some function of the nucleic acid (DNA orRNA) per se of the Rf-1 gene in conferring fertility.

Whether or not a homolog or variant of the Rf-1 gene functions torestore fertility can be examined by, but not limited to, the followingmethod, for example. A nucleic acid fragment under test is introducedinto immature seeds obtained by pollinating MS Koshihikari (sterileline) with MS-FR Koshihikari according to the method of Hiei et al.(Plant Journal (1994), 6(2), p. 272-282). As the resulting transformantsare cultured under normal conditions, the seeds mature only when thenucleic acid fragment under test functions to restore fertility.

The nucleic acid derived from a corresponding region of japonicaAsominori not carrying the Rf-1 gene has the base sequence shown in SEQID NO:28. Corresponding parts of SEQ ID NO:28 and SEQ ID NO:27 have anoverall identity of about 98%. Thus, nucleic acids containing the locusof a fertility restorer gene (Rf-1) of the present invention are atleast about 70%, preferably about 80% or more, more preferably 90% ormore, still more preferably 95% or more, most preferably 98 or more %identical to SEQ ID NO:27. Especially, the term “SEQ ID NO:27” intendsto mean any one of g) the bases 43907-46279 of SEQ ID NO:27, oralternatively, a) the bases 215-2587 of SEQ ID NO:69, b) the bases213-2585 of SEQ ID NO:70, c) the bases 218-2590 of SEQ ID NO:71, d) thebases 208-2580 of SEQ ID NO:72, e) the bases 149-2521 of SEQ ID NO:73,f) the bases 225-2597 of SEQ ID NO:74, h) the bases 229-2601 of SEQ IDNO:80, i) the bases 175-2547 of SEQ ID NO:81, j) the bases 227-2599 ofSEQ ID NO:82, k) the bases 220-2592 of SEQ ID NO:83, l) the bases174-2546 of SEQ ID NO:84 or m) the bases 90-2462 of SEQ ID NO:85corresponding thereto.

The percent identity of a nucleic acid may be determined by visualinspection and mathematical calculation. Alternatively, the percentidentity of two nucleic acid sequences can be determined by comparingsequence information using the GAP computer program, version 6.0described by Devereux et al., Nucl. Acids Res., 12:387 (1984) andavailable from the University of Wisconsin Genetics Computer Group(UWGCG). The preferred default parameters for the GAP program include:(1) a unary comparison matrix (containing a value of 1 for identitiesand 0 for non-identities) for bases, and the weighted comparison matrixof Gribskov and Burgess, Nucl. Acids Res., 14:6745 (1986), as describedby Schwartz and Dayhoff, eds., Atlas of Protein Sequence and Structure,National Biomedical Research Foundation, pp. 353-358 (1979); (2) apenalty of 3.0 for each gap and an additional 0.10 penalty for eachsymbol in each gap; and (3) no penalty for end gaps. Other programs usedby those skilled in the art of sequence comparison may also be used.

Nucleic acids of the present invention also include nucleic acids whichare capable of hybridizing to the base sequence of SEQ ID NO:27 underconditions of moderately stringent conditions and functions to restorefertility, and nucleic acids which are capable of hybridizing to thebase sequence of SEQ ID NO:27 under conditions of highly stringentconditions and functions to restore fertility.

As used herein, conditions of moderate stringency can be readilydetermined by those having ordinary skill in the art based on, forexample, the length of the DNA. The basic conditions are set forth bySambrook et al. Molecular Cloning: A Laboratory Manual, 2nd. Vol. 1, pp.1.101-104, Cold Spring Harbor Laboratory Press, (1989), and include useof a prewashing solution for the nitrocellulose filters 5×SSC, 0.5% SDS,1.0 mM EDTA (pH 8.0), hybridization conditions of about 1×SSC to 6×SSCat about 40° C. to 60° C. (or other similar hybridization solution, suchas Stark's solution, in about 50% formamide at about 42° C.), andwashing conditions of about 60° C., 0.5×SSC, 0.1% SDS. The hybridizationtemperature is about 15-20° C. lower when the hybridization solutioncontains about 50% formamide. Conditions of high stringency can also bereadily determined by the skilled artisan based on, for example, thelength of the DNA. Generally, conditions of high stringency includehybridization and/or washing conditions at higher temperatures and/orlower salt concentrations than in the conditions of moderate stringencydescribed above. For example, such conditions include hybridizationconditions of 0.1×SSC to 0.2×SSC at about 60-65° C. and/or washingconditions of 0.2×SSC, 0.1% SDS at about 65-68° C. The skilled artisanwill recognize that the temperature and wash solution salt concentrationcan be adjusted as necessary according to factors such as the length ofthe probe.

Especially preferably, “SEQ ID NO:27” intends to mean any one of g) thebases 43907-46279 of SEQ ID NO:27, or alternatively, a) the bases215-2587 of SEQ ID NO:69, b) the bases 213-2585 of SEQ ID NO:70, c) thebases 218-2590 of SEQ ID NO:71, d) the bases 208-2580 of SEQ ID NO:72,e) the bases 149-2521 of SEQ ID NO:73, f) the bases 225-2597 of SEQ IDNO:74, h) the bases 229-2601 of SEQ ID NO:80, i) the bases 175-2547 ofSEQ ID NO:81, j) the bases 227-2599 of SEQ ID NO:82, k) the bases220-2592 of SEQ ID NO:83, l) the bases 174-2546 of SEQ ID NO:84 or m)the bases 90-2462 of SEQ ID NO:85 corresponding thereto.

DNAs of the present invention also include nucleic acids that differfrom the base sequence of SEQ ID NO:27 due to deletions, insertions orsubstitutions of one or more bases while retaining a fertility restoringfunction. So far as a fertility restoring function is retained, thenumber of bases to be deleted, inserted or substituted is notspecifically limited, but preferably 1 to several thousands, morepreferably 1-1000, still more preferably 1-500, even more preferably1-200, most preferably 1-100.

The Rf-1 gene has further been specified on the basis of thedescriptions herein, and it can be used by those skilled in the artafter nucleic acids such as other regions than the Rf-1 gene or intronregions in the Rf-1 gene are removed. A given amino acid (especially,the amino acid sequence of SEQ ID NO:75) may be replaced, for example,by a residue having similar physicochemical characteristics. Examples ofsuch conservative substitutions include changes from one aliphaticresidue to another, such as changes from one to another of Ile, Val,Leu, or Ala; changes from one polar residue to another, such as changesbetween Lys and Arg, Glu and Asp, or Gln and Asn; or changes from onearomatic residue to another, such as changes from one to another of Phe,Trp, or Tyr. Other well-known conservative substitutions include e.g.changes between entire regions having similar hydrophobiccharacteristics. Those skilled in the art can introduce desireddeletions, insertions or substitutions by well-known gene engineeringtechniques using e.g. site-specific mutagenesis as described in Sambrooket al., Molecular Cloning: A Laboratory Manual, 2nd edition, Cold SpringHarbor Laboratory Press, (1989).

We compared an indica variety IR24 carrying the Rf-1 gene (SEQ ID NO:27)with japonica varieties not carrying it such as Asominori (SEQ ID NO:28)and a Nipponbare BAC clone deposited with GenBank (Accession No.AC068923). As a result, we found that the Rf-1 region of the indicavariety containing the Rf-1 gene has at least the following single basespolymorphisms (SNP).

1) a base corresponding to the base 1239 of SEQ ID NO:27 is A;

2) a base corresponding to the base 6227 of SEQ ID NO:27 is A;

3) a base corresponding to the base 20680 of SEQ ID NO:27 is G;

4) a base corresponding to the base 45461 of SEQ ID NO:27 is A;

5) a base corresponding to the base 49609 of SEQ ID NO:27 is A;

6) a base corresponding to the base 56368 of SEQ ID NO:27 is T;

7) a base corresponding to the base 57629 of SEQ ID NO:27 is C; and

8) a base corresponding to the base 66267 of SEQ ID NO:27 is G.

Thus, nucleic acids containing the Rf-1 region of the present inventionpreferably meet one to all of the requirements 1)-8) above.

In Example 3 below, the chromosomal organizations of recombinantsproximal to the Rf-1 gene (RS1-RS2, RC1-RC8) were tested in the Rf-1region. The results showed that a sequence determining the presence ofthe function of the Rf-1 gene is contained in the base sequence of bases1239-66267 of SEQ ID NO:27, i.e. in a region from the P4497 MboI toB56691 XbaI loci (about 65 kb) as estimated at maximum (FIG. 3).However, there is a possibility that it is important for the expressionof the genetic function of the Rf-1 gene that the Rf-1 gene is partiallyof the indica genotype, and that the genetic function may not besignificantly changed whether the remaining regions are of the japonicaor indica genotype. There may be an extreme case that the coding regionis completely identical and only the promoter region is differentbetween japonica and indica, and that the promoter region and the codingregion are only partially included in the region from P4497 the MboI toB56691 XbaI loci (about 65 kb). Therefore, it cannot be concluded thatthe common indica region above (bases 1239-66267 of SEQ ID NO:27)completely contains the entire Rf-1 gene. However, it is thought that atleast SEQ ID NO:27 completely contains the entire Rf-1 gene for thefollowing reasons:

1) the size of a gene is normally several kilobases, and rarely exceeds10 kb;

2) the genomic base sequence of IR24 determined by the present invention(SEQ ID NO:27) completely contains the common indica region above;

3) the 5′ end of SEQ ID NO:27 is located 1238 bp upstream of the 5′ endof the common indica region above and forms a part of another gene(S12564); and

4) the 3′ end of SEQ ID NO:27 is located 10096 bp downstream of the 3′end of the common indica region above.

In this way, we first succeeded in restricting the region of the Rf-1gene to 76 kb. Thus, nucleic acids containing the region of the Rf-1gene of the present invention are extremely less likely to contain othergenes proximal to the Rf-1 gene as compared with those selected with theco-dominant marker locus at a genetic distance of about 1 cM (about 300kb) from the Rf-1 gene described in a prior documents such as JapanesePatent Public Disclosure No. 2000-139465. Moreover, they are less likelyto contain other genes than those selected with the DNA marker lociS12564 Tsp509I and C1361 MwoI (at a distance of about 0.3 cM betweenthem) described in our prior Japanese Patent Application No.2000-247204.

We further confirmed by complementation assays that the Rf-1 gene iscompletely contained in especially bases 38538-54123 of the basesequence of SEQ ID NO:27. In an embodiment of the present invention,therefore, the base sequence at least 70% identical to the base sequenceof SEQ ID NO:27 or to the base sequence of bases 38538-54123 of SEQ IDNO:27 meets at least one of the following requirements 1) and 2):

1) a base corresponding to the base 45461 of SEQ ID NO:27 is A;

2) a base corresponding to the base 49609 of SEQ ID NO:27 is A.

The present inventors further determined that the following regions asbeing nucleic acids containing the Rf-1 gene.

a) the bases 215-2587 of SEQ ID NO:69;

b) the bases 213-2585 of SEQ ID NO:70;

c) the bases 218-2590 of SEQ ID NO:71;

d) the bases 208-2580 of SEQ ID NO:72;

e) the bases 149-2521 of SEQ ID NO:73;

f) the bases 225-2597 of SEQ ID NO:74;

h) the bases 229-2601 of SEQ ID NO:80;

i) the bases 175-2547 of SEQ ID NO:81;

j) the bases 227-2599 of SEQ ID NO:82;

k) the bases 220-2592 of SEQ ID NO:83;

l) the bases 174-2546 of SEQ ID NO:84; and

m) the bases 90-2462 of SEQ ID NO:85.

The above base sequences correspond to g) the bases 43907-46279 of SEQID NO:27. The nucleic acids of the present invention further include

n) a nucleic acid which is identical to at least 70% of the nucleic acidof any of a)-m), and which functions to restore fertility;

o) a nucleic acid which hybridizes to the nucleic acid of any of a)-m)under a moderate or high stringent condition, and which functions torestore fertility; and

p) a nucleic acid wherein one or a plurality of base(s) is deleted from,added to or substituted from the nucleic acid of any of a)-m), and whichfunctions to restore fertility.

The base 45461 of SEQ ID NO:27 corresponds to 1) the base 1769 of SEQ IDNO. 69; 2) the base 1767 of SEQ ID NO. 70; 3) the base 1772 of SEQ IDNO. 71; 4) the base 1762 of SEQ ID NO. 72; 5) the base 1703 of SEQ IDNO. 73; 6) the base 1779 of SEQ ID NO. 74; 7) the base 1783 of SEQ IDNO. 80; 8) the base 1729 of SEQ ID NO. 81; 9) the base 1781 of SEQ IDNO. 82; 10) the base 1774 of SEQ ID NO. 83; 11) the base 1728 of SEQ IDNO. 84; and 12) the base 1644 of SEQ ID NO. 85. Accordingly, especiallypreferably, the nucleic acid used for the method of the presentinvention meets at least one of the following requirements 1)-12):

1) a base corresponding to the base 1769 of SEQ ID NO. 69 is A;

2) a base corresponding to the base 1767 of SEQ ID NO. 70 is A;

3) a base corresponding to the base 1772 of SEQ ID NO. 71 is A;

4) a base corresponding to the base 1762 of SEQ ID NO. 72 is A;

5) a base corresponding to the base 1703 of SEQ ID NO. 73 is A;

6) a base corresponding to the base 1779 of SEQ ID NO. 74 is A;

7) a base corresponding to the base 1783 of SEQ ID NO. 80 is A;

8) a base corresponding to the base 1729 of SEQ ID NO. 81 is A;

9) a base corresponding to the base 1781 of SEQ ID NO. 82 is A;

10) a base corresponding to the base 1774 of SEQ ID NO. 83 is A;

11) a base corresponding to the base 1728 of SEQ ID NO. 84 is A; or

12) a base corresponding to the base 1644 of SEQ ID NO. 85 is A.

IV. Method for Restoring Rice Fertility

The present invention provides a method for restoring rice fertilitycomprising introducing a nucleic acid into rice, wherein the nucleicacid has the base sequence of SEQ ID NO. 27, or has a base sequencewhich is identical to at least 70% of the base sequence of SEQ ID NO.27, and which functions to restore fertility. The methods of the presentinvention may comprise introducing a nucleic acid into rice, wherein thenucleic acid has a portion of SEQ ID NO:27, especially the bases38538-54123, preferably the bases 42357-53743, more preferably the bases42132-48883 of SEQ ID NO:27 or has a base sequence which is at least 70%identical to the base sequence of bases 38538-54123, preferably thebases 42357-53743, more preferably the bases 42132-48883 of SEQ IDNO:27, still more preferably the bases 42132-46318 and, which functionsto restore fertility.

In a particularly preferable embodiment of the present method, thenucleic acid encodes the amino acid sequence of SEQ ID NO. 75, or anamino acid sequence which is identical to at least 70% of the amino acidsequence of SEQ ID NO. 75, and which functions to restore fertility isintroduced into rice. Most preferably, the nucleic acid encoding theamino acid sequence of SEQ ID NO. 75, or an amino acid sequence which isidentical to at least 70% of the amino acid sequence of SEQ ID NO. 75 isselected from nucleic acids of the following a)-p):

a) a nucleic acid comprising the bases 215-2587 of SEQ ID NO:69;

b) a nucleic acid comprising the bases 213-2585 of SEQ ID NO:70;

c) a nucleic acid comprising the bases 218-2590 of SEQ ID NO:71;

d) a nucleic acid comprising the bases 208-2580 of SEQ ID NO:72;

e) a nucleic acid comprising the bases 149-2521 of SEQ ID NO:73;

f) a nucleic acid comprising the bases 225-2597 of SEQ ID NO:74;

g) a nucleic acid comprising the bases 43907-46279 of SEQ ID NO:27;

h) a nucleic acid comprising the bases 229-2601 of SEQ ID NO:80;

i) a nucleic acid comprising the bases 175-2547 of SEQ ID NO:81;

j) a nucleic acid comprising the bases 227-2599 of SEQ ID NO:82;

k) a nucleic acid comprising the bases 220-2592 of SEQ ID NO:83;

l) a nucleic acid comprising the bases 174-2546 of SEQ ID NO:84;

m) a nucleic acid comprising the bases 90-2462 of SEQ ID NO:85;

n) a nucleic acid which is identical to at least 70% of the nucleic acidof any of a)-m), and which functions to restore fertility;

o) a nucleic acid which hybridizes to the nucleic acid of any of a)-m)under a moderate or high stringent condition, and which functions torestore fertility; and

p) a nucleic acid wherein one or a plurality of base(s) is deleted from,added to or substituted from the nucleic acid of any of a)-m), and whichfunctions to restore fertility.

In the present invention, the nucleic acid containing the locus of afertility restorer gene (Rf-1) that can be introduced into rice can beany one of the nucleic acids described above in “III. Nucleic acidscontaining the Rf-1 locus”. The method for introducing the nucleic acidinto rice is not specifically limited but can be any known method.Nucleic acids of the present invention can be introduced by knowngenetic engineering techniques or crossing. Genetic engineeringtechniques are preferably used because inclusion of other neighboringgenes can be prevented and the period for establishing a line can beshortened.

Any suitable expression system for transduction by genetic engineeringtechniques can be employed. Recombinant expression vectors comprise anucleic acid containing a fertility restorer gene (Rf-1) of theinvention that can be introduced into rice, operably linked to suitabletranscriptional or translational regulatory base sequences, such asthose derived from a mammalian, microbial, viral, or insect gene.

Examples of regulatory sequences include transcriptional promoters,operators, or enhancers, an mRNA ribosomal binding site, and appropriatesequences which control transcription and translation initiation andtermination. Base sequences are operably linked to a regulatory sequencewhen the regulatory sequence is functionally associated with the DNAsequences. Thus, a promoter base sequence is operably linked to a DNAsequence if the promoter base sequence controls the transcription of theDNA sequence. An origin of replication that confers the ability toreplicate in rice, and a selection gene by which transformants areidentified, are generally incorporated into expression vectors. As forselectable markers, those commonly used can be used by standard methods.Examples are genes resistant to antibiotics such as tetracycline,ampicillin, kanamycin, neomycin, hygromycin or spectinomycin.

In addition, a sequence encoding an appropriate signal peptide (nativeor heterogonous) can be incorporated into expression vectors. A DNAsequence for a signal peptide (secretary leader) may be fused in frameto a nucleic acid sequence of the invention so that the DNA is initiallytranscribed, and the mRNA translated into a fusion protein containingthe signal peptide.

The present invention also provides recombinant vectors containing agene of the present invention. Methods for integrating a DNA fragment ofa gene of the present invention into a vector such as a plasmid aredescribed in e.g. Sambrook, J. et al, Molecular Cloning, A LaboratoryManual (2nd edition), Cold Spring Harbor Laboratory, 1.53 (1989).Commercially available ligation kits (e.g. available from TAKARA) can beconveniently used. Thus obtained recombinant vectors (e.g. recombinantplasmids) are transferred into host rice cells.

Vectors can be conveniently prepared by linking a desired gene to arecombinant vector available in the art (e.g. plasmid DNA) by standardmethods. Plant transforming vectors are especially useful for conferringfertility on rice using a nucleic acid fragment of the presentinvention. Vectors for plants are not specifically limited so far asthey can express the gene of interest in plant cells to produce theprotein, but preferably include pBI221, pBI121 (Clutch), and vectorsderived there from. Especially, examples of vectors for transformingrice belonging to monocotyledons include pIG121Hm and pTOK233 (Hiei etal., Plant J., 6, 271-282 (1994)), and pSB424 (Komari et al., Plant J.,10, 165-174 (1996)).

Transgenic plants can be prepared by replacing the β-glucuronidase (GUS)gene in the above vectors with a nucleic acid fragment of the presentinvention to construct a plant transforming vector and transfecting itinto a plant. The plant transforming vector preferably comprises atleast a promoter, a start codon, a desired gene (a nucleic acid sequenceof the present invention or a part thereof), a stop codon and aterminator. It may also contain a DNA encoding a signal peptide, anenhancer sequence, non-translated 5′ and 3′ regions of the desired gene,a selectable marker region, etc., as appropriate. Promoters andterminators are not specifically limited so far as they are functionalin plant cells, among which constitutive expression promoters includethe 35S promoter initially contained in the above vectors as well aspromoters for actin and ubiquitin genes.

Suitable methods for introducing a plasmid into a host cell include theuse of calcium phosphate or calcium chloride/rubidium chloride,electroporation, electroinjection, chemical treatment with PEG or thelike, the use of a gene gun described in Sambrook, J. et al., MolecularCloning, A Laboratory Manual (2nd edition), Cold Spring HarborLaboratory, 1.74 (1989). Plant cells can be transformed by e.g. the leafdisc method [Science, 227, 129 (1985)] or electroporation [Nature, 319,791 (1986)].

Methods for transferring a gene into a plant include the use ofAgrobacterium (Horsch et al., Science, 227, 129 (1985); Hiei et al.,Plant J., 6, 271-282 (1994)), electroporation (Fromm et al., Nature,319, 791 (1986)), PEG (Paszkowski et al., EMBO J., 3, 2717 (1984)),microinjection (Crossway et al., Mol. Gen. Genet., 202, 179 (1986)),particle bombardment (McCabe et al., Bio/Technology, 6, 923 (1988)).Methods are not specifically limited so far as they are suitable fortransfecting a nucleic acid into a desired plant.

Transduction by crossing can be performed as follows, for example.First, F₁ obtained by crossing an Rf-1 donor parent and a japonicavariety is backcrossed with the japonica variety. The resultingindividuals are screened for those homozygous for japonica at the S12564Tsp509I locus and heterozygous at the P4497 MboI and B53627 BstZ17I lociand further backcrossed. The resulting individuals are screened forthose heterozygous at the P4497 MboI and B56691 XbaI loci and homozygousfor japonica at the B53627 BstZ17I locus and further backcrossed.Subsequently, about 10 cycles of screening each backcrossed generationfor individuals heterozygous at the P4497 MboI and B56691 XbaI loci andsubjecting them to the subsequent backcrossing are repeated. Finally,individuals heterozygous at the P4497 MboI and B56691 XbaI loci areself-fertilized and the resulting individuals are screened for thosehomozygous for indica at both loci, whereby a restorer line inheriting alimited chromosomal region from the P4497 MboI to B56691 XbaI loci fromthe Rf-1 donor parent can be obtained.

According to the present invention, nucleic acids containing a fertilityrestorer gene (Rf-1) were isolated, whereby the Rf-1 gene can beintroduced into a rice variety using genetic engineering techniques toestablish a restorer line. The present invention succeeded inrestricting the Rf-1 region to 76 kb or less in the first place.Therefore, nucleic acids containing the Rf-1 locus of the presentinvention are extremely less likely to contain other genes neighboringthe Rf-1 gene than those of the prior art. Moreover, the entire basesequence of the region containing the Rf-1 gene was determined by thepresent invention. Those skilled in the art can proceed with analysis ofthe Rf-1 gene itself on the basis of the description herein. Thus, onlythe Rf-1 gene can be introduced without including any neighboring gene.This is especially important when neighboring genes bring deleterioustraits. Furthermore, restorer lines can be established in a shorterperiod such as 1-2 years than obtained by crossing.

In complementation assays described in Examples 4-13 and 17 herein, MSKoshihikari (having BT cytoplasm and a core gene substantially identicalto Koshihikari) was actually transformed by an Agrobacterium-mediatedmethod using fragments from 10 clones described in FIG. 5. The resultsdemonstrated that fertility restorer lines can be established from anucleic acid containing the base sequence of the bases 38538-54123,preferably the bases 42357-53743, more preferably the bases 42132-48883,still more preferably the bases 42132-46318 of SEQ ID NO:27.

Agrobacterium-mediated methods for establishing rice restorer lines aredescribed in, but not limited to, Hiei et al., Plant J., 6, pp. 271-282(1994), Komari et al., Plant J., 10, p. 165-174 (1996), Ditto et al.,Proc. Natl. Acad. Sci. USA 77: pp. 7347-7351 (1980), etc.

First, a plasmid vector containing a nucleic acid fragment of interestto be inserted is prepared. Suitable plasmid vectors include e.g. pSB11,pSB22 and the like having a plasmid map described in Komari et al.,Plant J., 10, pp. 165-174 (1996), supra. Alternatively, those skilled inthe art can also construct an appropriate vector by themselves on thebasis of plasmid vectors such as pSB11, pSB22 described above. In theexamples herein below, an intermediate vector pSB200 having ahygromycin-resistant gene cassette was prepared on the basis of pSB11,and used. Specifically, a nonaligned syntheses terminator (Tons) wasfirst fused to a ubiquitin promoter and a ubiquitin intron (Pubi-ubiI).A hygromycin-resistant gene (HYG(R)) was inserted between ubiI and Tonsof the resulting Pubi-ubiI-Tnos complex to give a Pubi-ubiI-HYG(R)-Tonsassembly. This assembly was fused to a HindIII/EcoRI fragment of pSB11(Komari et al., supra.) to give pKY205. Linker sequences for addingrestriction enzyme sites NotI, NspV, EcoRV, KpnI, SacI, EcoRI wereinserted into the Hind III site upstream of Pubi of this pKY205 to givepSB200 having a hygromycin-resistant gene cassette.

Then, E. coli cells (e.g. DH5a, JM109, MV1184, all commerciallyavailable from e.g. TAKARA) are transformed with the recombinant vectorcontaining the nucleic acid inserted.

Thus transformed E. coli cells are used for triparental mating with anAgrobacterium strain preferably in combination with a helper E. colistrain according to e.g. the method of Ditto et al. (1980). SuitableAgrobacterium strains include Agrobacterium tumefaciens strains such asLBA4404/pSB1, LBA4404/pNB1, LBA4404/pSB3, etc. They all have a plasmidmap described in Komari et al., Plant J., 10, pp. 165-174 (1996), supra.and can be used by those skilled in the art by constructing a vector bythemselves. Suitable helper E. coli strains include, but not limited to,e.g. HB101/pRK2013 (available from Clutch). A report shows that E. colicells carrying pRK2073 can also be used as helper E. coli though theyare less common (Lemas et al., Plasmid 1992, 27, pp. 161-163).

Then, the Agrobacterium cells mated as intended are transformed intomale sterility rice according to e.g. the method of Hiei et al (1994).Necessary immature rice seeds for transformation can be prepared by e.g.pollinating male sterility rice with a japonica variety.

Fertility restoration in transformed plants can be assessed by e.g.evaluating seed fertility in standing plants about one month afterheading. Evaluation on standing plants means observation of plants grownin a field or the like. An alternative method is a laboratory study ofgrain ripening percentages in the ear.

V. Methods for Discerning the Presence of the Rf-1 Gene

According to the present invention, it was shown that a sequencedetermining the presence of the function of the Rf-1 gene is locatedbetween the polymorphism-detecting marker loci P4497 MboI and B56691XbaI on rice chromosome 10. Moreover, complementation assays confirmedthat the Rf-1 gene is completely contained in especially bases38538-54123 of the base sequence of SEQ ID NO:27.

Comparison of the base sequence of an indica variety carrying the Rf-1gene (IR24) (SEQ ID NO:27) with those of japonica varieties not carryingsaid gene (Asominori (SEQ ID NO:28) and Nipponbare BAC clone AC068923)revealed the presence of polymorphisms between both varieties. As aresult, it became possible to conveniently, rapidly and exactly discernwhether or not a rice plant or seed under test carries the Rf-1 gene onthe basis of polymorphisms in base sequence in regions neighboring theRf-1 gene.

Therefore, the present invention also provides a method for discerningwhether or not a subject rice individual or a seed thereof has the Rf-1gene or not, wherein the method utilizing a fact that a sequencedetermining the presence of the function of the Rf-1 gene positionsbetween the polymorphism detection marker loci P4497 MboI and B56691 XbaI on rice chromosome 10.

Polymorphisms can be detected by any known method. For example, knownmethods include assays for restriction fragment length polymorphisms(RFLPs); direct determination by sequencing; cutting a genomic DNA witha 8-base recognizing restriction enzyme, and then radioactively labelingthe ends and further cutting the labeled digest with 6-base and 4-basesrecognizing restriction enzyme and then developing the digest bytwo-dimensional electrophoresis (RLGS, Restriction Landmark GenomeScanning); etc. AFLP analysis (amplified fragment length polymorphism;P. Vos et al., Nucleic Acids Res. Vol. 23, pp. 4407-4414 (1995)) hasalso been developed wherein RFLP is amplified/detected by polymerasechain reaction (PCR).

For example, conventional methods involved detecting RFLPs via PCRamplification (conversion of RFLP markers into PCR markers) or detectingpolymorphisms in microsatellites via PCR amplification (microsatellitemarkers) as illustrated below.

Conversion of RFLP Markers into PCR Markers

A. PCR markers based on polymorphisms in genomic regions correspondingto RFLP probes (D. E. Harry, B. Temesgen, D. B. Neale; CodominantPCR-based markers for Pinus taeda developed from mapped cDNA clones,Theor. Appl. Genet. (1998) 97: pp. 327-336). After performing genomicPCR using primers designed for an RFLP marker probe sequence (“RFLP” isa polymorphism observed by Southern analysis using a DNA fragment as aprobe. The base sequence of the DNA fragment used as a probe is called“RFLP marker probe sequence”), a PCR marker can be prepared by either ofthe following two procedures. A first procedure involves treating theproducts with a series of restriction enzymes to search for arestriction enzyme causing a fragment length polymorphism, and a secondprocedure involves searching for a polymorphism by varietal comparisonof the base sequences of the products and preparing a PCR marker basedon the polymorphism.

B. PCR markers based on identification of RFLP-causing sites. A PCRmarker can be obtained by identifying an RFLP-causing site (arestriction enzyme recognition site carried by only one of two varietiescompared) present in or near (normally within several kbs) an RFLPmarker probe sequence.

Microsatellite Markers

Microsatellites are repeat sequences of about 2 to 4 bases such as(CA)_(n) that are present in great numbers in genomes. If a varietalpolymorphism occurs in repetition number, a polymorphism can be observedin PCR product length by PCR using primers designed in adjacent regions,whereby the DNA polymorphism can be detected. Markers for detectingpolymorphisms using microsatellites are called microsatellite markers(O. Parnaud, X. Chen, S. R. McCouch, Mol. Gen. Genet. (1996) 252: pp.597-607).

Methods for detecting polymorphisms in the present invention are notspecifically limited. From the viewpoint of efficiency and convenience,PCR-RFLP is preferred, which is a combination of PCR and RFLP toidentify polymorphisms from their restriction enzyme cleavage patternsin cases where they exist among variety lines at restriction enzymerecognition sites in the sequences of DNA fragments amplified by PCR.PCR-RFLP is also called CAPS (cleaved amplified polymorphic sequence).If any suitable restriction enzyme recognition site is not present in aregion showing polymorphisms, a modified CAPS called dCAPS (derivedcleaved amplified polymorphic sequence) can also be used whereinrestriction enzyme sites are introduced during PCR (Michaels, S. D. andAmasino, R. M. (1998), The Plant Journal 14(3) 381-385; A. Konieczny etal., (1993), Plant J. 4(2) pp. 403-410; Neff, M. M., Neff, J. D., Chory,J. and Pepper, A. E. (1998), The Plant Journal 14(3) 387-392). Thesemethods are explained in more detail below.

CAPS, dCAPS

The method for discerning of the present invention comprise, but notlimited to:

i) preparing a pair of primers based on the base sequences of a siteshowing a polymorphism in the base sequences between indica and japonicavarieties at the Rf-1 locus and its adjacent regions to amplify saidbase sequences;

ii) performing nucleic acid amplification reaction(s) using the genomicDNA of the subject rice individual or the seed thereof as a template;and

iii) discerning whether or not the subject rice individual or the seedthereof has the Rf-1 gene based on the polymorphism found in the nucleicacid amplification product.

The step of preparing a primer pair in i) preferably comprises any ofthe following means:

a) when a change containing a deleted region exists in the polymorphismin the nucleic acid amplification product, preparing a pair of primersfor nucleic acid amplification to flank the deleted region to form amarker for detecting the polymorphism;

b) when a base change causing a difference in restriction enzymerecognition exists in the polymorphism in the nucleic acid amplificationproduct, preparing a pair of primers for nucleic acid amplification toflank the base change site to form a marker for detecting thepolymorphism; or

c) when a base change causing no difference in restriction enzymerecognition exists in the polymorphism in the nucleic acid amplificationproduct, preparing a pair of primers for introducing a mismatch, whereinpair of primers contain the base change site and alters a regioncontaining the base change site into a base sequence causing adifference in restriction enzyme recognition in the nucleic acidamplification product to form a marker for detecting the polymorphism.

Suitable polymorphic sites for discerning the presence of the Rf-1 genein the present invention can be appropriately selected so that apolymorphism detecting marker can be prepared as described below on thebasis of comparison of, but not limited to, the base sequence of anindica variety carrying the Rf-1 gene (IR24) (SEQ ID NO:27) with thoseof japonica varieties not carrying said gene (Asominori (SEQ ID NO:28)and Nipponbare BAC clone AC068923).

If the polymorphism found causes a difference in restriction enzymerecognition, for example, a pair of primers for nucleic acidamplification are prepared to flank the polymorphic site and used fordetecting the polymorphism. Primers are preferably designed not to bespecific for highly repeated sequences to avoid undesired products. Ifthe polymorphism found does not cause a difference in restriction enzymerecognition, markers can be prepared by applying the dCAPS methoddescribed above. Primers for dCAPS markers are preferably designed notto be specific for repeat sequences and to provide a product length ofpreferably 50-300 bases, more preferably about 100 bases to easeidentification of polymorphisms.

If the polymorphism found involves a microsatellite, nucleic acidamplification primers are prepared to flank the microsatellite and usedto detect the polymorphism. Again, the primers are preferably designednot to be specific for repeat sequences.

1) Nucleic Acid Amplification

In the present invention, a pair of primers are preferably prepared foramplifying adjacent regions containing polymorphisms on the basis of thedetermined base sequence of the nucleic acid of a subject riceindividual or seed at the Rf-1 locus. The primer pair is used to performa nucleic acid amplification reaction with the genomic DNA of thesubject rice individual or seed as a template. The nucleic acidamplification reaction is preferably polymerase chain reaction (PCR)(Saiki et al., 1985, Science 230, pp. 1350-1354).

The pair of primers for nucleic acid amplification can be prepared byany known method on the basis of the base sequence of a polymorphic siteand adjacent regions thereto. Specifically, a primer pair can beprepared on the basis of the base sequence of a polymorphic site andadjacent regions thereto by a process comprising generating asingle-stranded DNA having the same base sequence as the base sequenceof the polymorphic site and adjacent regions thereto or a base sequencecomplementary to said regions or, if necessary, generating thesingle-stranded DNA containing a modification without affecting thebinding specificity to the base sequence of the polymorphic site andadjacent regions thereto provided that the following conditions aresatisfied:

1) the length of each primer should be 15-30 bases;

2) the proportion of G+C in the base sequence of each primer should be30-70%;

3) the distribution of A, T, G and C in the base sequence of each primershould not be partially largely uneven;

4) the length of the nucleic acid amplification product amplified by theprimer pair should be 50-3000 bases, preferably 50-300 bases; and

5) any complementary sequence segment should not occur with the basesequence of each primer or between the base sequences of the primers.

As used herein, the “adjacent regions” to a polymorphic site mean thatan area containing both of a polymorphic site and adjacent regionsthereto is within a distance suitable for nucleic acid amplification,preferably PCR. The adjacent regions amplified preferably have a lengthwithin the range of, but not limited to, about 50 bases to about 3000bases, more preferably about 50 bases to about 2000 bases. To facilitateidentification of polymorphisms, the product length is preferably 50-300bases, more preferably about 100 bases. The adjacent regions preferablyhave a length within the range of, but not limited to, about 0 to about3000 bases, more preferably about 0 to about 2000 bases, still morepreferably about 0 to about 1000 bases on the 5′ or 3′ side of apolymorphic site.

Procedures and conditions for the nucleic acid amplification reactionare not specifically limited and are well known to those skilled in theart. Appropriate conditions can be applied by those skilled in the artdepending on various factors such as the base sequence of thepolymorphic site and adjacent regions thereto, the base sequence andlength of the primer pair, etc. Generally, the nucleic acidamplification reaction can be performed under more stringent conditions(annealing reaction and nucleic acid elongation reaction at highertemperatures and less cycles) as the primer pair is longer or theproportion of G+C is higher or the distribution of A, T, G and C isevener. The use of more stringent conditions allows an amplificationreaction with higher specificity.

The amplification reaction can be performed under conditions of, but notlimited to, one cycle of 94° C. for 2 min, 30 cycles of 94° C. for 1min, 58° C. for 1 min and 72° C. for 2 min, and finally one cycle of 72°C. for 2 min using 50 ng of a genomic DNA as a template, 200 μM of eachdNTP and 5 U of ExTaq™ (TAKARA). The reaction can also be performedunder conditions of one cycle of 94° C. for 2 min, 30 cycles of 94° C.for 1 min, 58° C. for 1 min and 72° C. for 1 min, and finally one cycleof 72° C. for 2 min. In another embodiment, the reaction can also beperformed under conditions of one cycle of 94° C. for 2 min, 35 cyclesof 94° C. for 30 sec, 58° C. for 30 sec and 72° C. for 30 sec, andfinally one cycle of 72° C. for 2 min.

The subject rice (test rice) genomic DNA used as a template for PCR canbe easily extracted from individuals or seeds by the method of Edwardset al. (Nucleic Acids Res. 8(6):1349, 1991). More preferably, DNApurified by standard techniques is used. An especially preferredextraction method is the CTAB method (Murray, M. G. et al., NucleicAcids Res. 8(19):4321-5, 1980). The DNA is preferably used as a templatefor PCR at a final concentration of 0.5 ng/μL.

2) Preparation of Markers for Detecting Polymorphisms

After examining whether or not a polymorphism is detected in theamplification product by the nucleic acid amplification reaction with apair of primers, a marker for detecting the polymorphism is prepared onthe basis of the polymorphism found. Non-limiting examples ofpolymorphisms that can be detected in the amplification product are asfollows.

a) A change containing a deleted region exists in the polymorphism inthe nucleic acid amplification product.

In this case, a pair of primers for nucleic acid amplification areprepared to flank the deleted region to form a marker for detecting thepolymorphism. If the deleted region has a sufficient size, thepolymorphism can be detected from the difference in mobility byelectrophoresing the amplification product on an agarose gel or anacrylamide gel, for example. The polymorphism can be detected when thedifference in base pair numbers is about 5% or more in the case ofagarose gel electrophoresis or when the difference in length is about 1base or more in the case of sequencing acrylamide gel electrophoresis,for example. Alternatively, the polymorphism can be detected byhybridizing the nucleic acid amplification product using an oligobase ora DNA fragment having a complementary sequence to the base sequenceexcluding the deleted region as an analytical probe. Alternatively, thepolymorphism can be confirmed by determining the base sequence of theamplification product, if desired. Known techniques for electrophoresisof nucleic acids, hybridization, sequencing and the like can be used asappropriate by those skilled in the art. In this case, the difference inthe length of the amplification product directly reflects thepolymorphism and markers for detecting polymorphisms on this basis arecalled ALP (amplicon length polymorphism) markers.

b) A base change causing a difference in restriction enzyme recognitionexists in the polymorphism in the nucleic acid amplification product.

In this case, a pair of primers for nucleic acid amplification areprepared to flank the base change site to form a marker for detectingthe polymorphism. In this case, a base change causing a difference inrestriction enzyme recognition occurs in the polymorphism of the nucleicacid amplification product, i.e. the nucleic acid amplification productmay be cleaved or not with one or more specific restriction enzymes.Thus, the amplification product can be treated with the restrictionenzymes and electrophoresed on e.g. an agarose gel to detect thepolymorphism from the difference in mobility. The polymorphism can beconfirmed by determining the base sequence of the amplification product,if desired.

In this case, the difference in the length of the restriction fragmentof the amplification product by PCR or the like reflects thepolymorphism and markers for detecting polymorphisms on this basis arecalled CAPS markers or PCR-RFLP markers (A. Konieczny et al., supra.)

This is exemplified by primer pairs P4497 MboI, P23945 MboI, P41030TaqI, P45177 BstUI, B59066 BsaJI and B56691 XbaI in Example 1 below.Even if the polymorphism can be detected by the length of the nucleicacid amplification product as described in a) above, the polymorphismcan be more easily detected by combination with restriction enzymetreatment.

c) A base change causing no difference in restriction enzyme recognitionexists in the polymorphism in the nucleic acid amplification product.

In this case, a pair of primers for introducing a mismatch are preparedthat contains the base change site and alters a region containing thebase change site into a base sequence causing a difference inrestriction enzyme recognition in the nucleic acid amplification productto form a marker for detecting the polymorphism.

Specifically, a pair of primers based on the base sequences of regionsnaturally proximal to the Rf-1 gene cause a polymorphism in the nucleicacid amplification product but no difference in restriction enzymerecognition, and therefore, a mismatch is introduced into one or both ofthe primers to alter a region containing the base change site(polymorphism) into a base sequence causing a difference in restrictionenzyme recognition in the nucleic acid amplification product. Forexample, the method described in Mikaelian et al., Nucl. Acids. Res.20:376. 1992 can be used as a standard technique for substituting,deleting or adding a specific base by PCR-mediated site-specificmutagenesis. The amplification product using the mismatch-introducingprimers as a marker for detecting the polymorphism may be cleaved or notwith one or more specific restriction enzymes because it has adifference in restriction enzyme recognition at the mismatch-introducingsite. Therefore, the amplification product can be treated with therestriction enzymes and electrophoresed on e.g. an agarose gel to detectthe polymorphism from the difference in mobility, as described in b)above.

The introduction of a mismatch must not affect not only the binding ofthe primers to a target plant genome but also the polymorphic basechange. The polymorphic base change is used to introduce a mismatch nearit so that a difference in restriction enzyme recognition occurs by acombination of both base change and mismatch. Methods for introducingsuch a mismatch are known to those skilled in the art and described indetail in Michaels, S. D. and Amasino, R. M. (1998), Neff, M. M., Neff,J. D., Chory, J. and Pepper, A. E. (1998), for example.

Markers in this case are improved CAPS markers described in b) above andcalled dCAPS (derived CAPS) markers. This is exemplified by P9493 BslIin Example 3 below.

If there are many extra restriction sites unrelated to varietalpolymorphisms in the case of b) or c) above, it may be difficult todiscern any difference in restriction site recognition based onpolymorphisms. In this case, a mismatch may be introduced into a primeras appropriate to abolish unnecessary restriction sites. For example, amismatch was introduced into the R-primer to abolish the MspI siteunrelated to polymorphisms in B60304 MspI in Example 3.

Although the invention is not limited to any specific method, CAPS ordCAPS methods have several advantages over other RFLP methods.Specifically, analyses can be made with smaller amounts of samples thanin RFLP, for example. Another advantage is that the time and laborrequired for analyses can be greatly reduced. Polymorphisms detectedwith PCR markers can be visualized by agarose gel electrophoresis thatis easier than acrylamide gel electrophoresis used for microsatellitemarkers.

Preferred Embodiments of the Discerning Method of the Present Invention

Preferred embodiments of the method for discerning whether or not asubject rice has the Rf-1 gene are described below for illustrativepurposes. In the examples herein, it was found that the base sequence ofan indica variety IR24 carrying the Rf-1 gene (SEQ ID NO:27) has atleast the following polymorphisms 1)-8) as compared with correspondingregions of japonica varieties:

1) a base corresponding to the base 1239 of SEQ ID NO:27 is A;

2) a base corresponding to the base 6227 of SEQ ID NO:27 is A;

3) a base corresponding to the base 20680 of SEQ ID NO:27 is G;

4) a base corresponding to the base 45461 of SEQ ID NO:27 is A;

5) a base corresponding to the base 49609 of SEQ ID NO:27 is A;

6) a base corresponding to the base 56368 of SEQ ID NO:27 is T;

7) a base corresponding to the base 57629 of SEQ ID NO:27 is C; and

8) a base corresponding to the base 66267 of SEQ ID NO:27 is G.

In preferred embodiments of the present invention, therefore, thesubject rice individual or seed is judged as carrying the Rf-1 gene whenone to all of the requirements 1)-8) above are met.

We further verified that a region essential for the expression of thefunction of the Rf-1 gene is contained in especially the bases38538-54123, preferably the bases 42357-53743, more preferably the bases42132-48883, still more preferably the bases 42132-46318 in the basesequence of SEQ ID NO:27. In an embodiment of the present invention,therefore, the subject rice individual or seed is determined to have theRf-1 gene in the case that the nucleic acid having a base sequence whichis identical to at least 70% of the base sequence of SEQ ID NO. 27 or ofthe base sequence of bases 38538-54123 of SEQ ID NO. 27, meets at leastone of the following requirements 1) and 2):

1) a base corresponding to the base 45461 of SEQ ID NO. 27 is A; and

2) a base corresponding to the base 49609 of SEQ ID NO. 27 is A.

Known polymorphism detecting methods can be used to determine whether ornot the above requirements are met. The base sequence of adjacentregions containing said sequence can also be directly determined.However, CAPS or dCAPS methods described above are preferably usedbecause they are rapid and convenient. CAPS or dCAPS methods can beperformed by a protocol comprising, for example:

i) preparing a pair of primers based on a base sequence of adjacentregions including any one of the following base;

1) a base corresponding to the base 1239 of SEQ ID NO:27;

2) a base corresponding to the base 6227 of SEQ ID NO:27;

3) a base corresponding to the base 20680 of SEQ ID NO:27;

4) a base corresponding to the base 45461 of SEQ ID NO:27;

5) a base corresponding to the base 49609 of SEQ ID NO:27;

6) a base corresponding to the base 56368 of SEQ ID NO:27;

7) a base corresponding to the base 57629 of SEQ ID NO:27; and

8) a base corresponding to the base 66267 of SEQ ID NO:27 is G.

to amplify both the base of the above and adjacent regions thereto;

ii) performing nucleic acid amplification reaction(s) using the genomeDNA of the subject rice individual or the seed thereof as a template;and

iii) discerning the presence of the Rf-1 in the subject rice individualor the seed thereof based on polymorphism found in said nucleic acidamplification product.

The detection of polymorphisms in the nucleic acid amplification productis performed by, but not limited to, discerning the subject riceindividual or seed to have the Rf-1 gene when one to all of therequirements 1)-8) below are met:

1) a region including a base corresponding to the base 1239 of SEQ IDNO:27 does not have any MboI recognition sequence;

2) a region including a base corresponding to the base 6227 of SEQ IDNO:27 does not have any BslI recognition sequence;

3) a region including a base corresponding to the base 20680 of SEQ IDNO:27 does not have any MboI recognition sequence;

4) a region including a base corresponding to the base 45461 of SEQ IDNO:27 does not have any TaqI recognition sequence;

5) a region including a base corresponding to the base 49609 of SEQ IDNO:27 does not have any BstUI recognition sequence;

6) a region including a base corresponding to the base 56368 of SEQ IDNO:27 does not have any MspI recognition sequence;

7) a region including a base corresponding to the base 57629 of SEQ IDNO:27 does not have any BsaJI recognition sequence; and

8) a region including a base corresponding to the base 66267 of SEQ IDNO:27 does not have any XbaI recognition sequence.

However, the present invention is not limited to the restriction enzymesabove so far as each polymorphism in the specific regions 1)-8) abovecan be detected.

Preferably, identification methods of the present invention comprise:

i) preparing a pair of primers based on a base sequence of adjacentregions including any one of the following base;

1) a base corresponding to the base 45461; or

2) a base corresponding to the base 49609;

to amplify both the base of the above and adjacent regions thereto;

ii) performing nucleic acid amplification reaction(s) using the genomeDNA of the subject rice individual or the seed thereof as a template;and

iii) discerning the presence of the Rf-1 in the subject rice individualor the seed thereof based on polymorphism found in said nucleic acidamplification product. The subject rice individual or seed thereof isdetermined to have the Rf-1 gene in step iii), although not limited to,when at least one of the following requirements 1) and 2) is met:

1) a region including a base corresponding to the base 45461 of SEQ IDNO:27 does not have any TaqI recognition sequence;

2) a region including a base corresponding to the base 49609 of SEQ IDNO:27 does not have any BstUI recognition sequence.

The base 45461 of SEQ ID NO:27 discussed above corresponds to 1) thebase 1769 of SEQ ID NO. 69; 2) the base 1767 of SEQ ID NO. 70; 3) thebase 1772 of SEQ ID NO. 71; 4) the base 1762 of SEQ ID NO. 72; 5) thebase 1703 of SEQ ID NO. 73; 6) the base 1779 of SEQ ID NO. 74; 7) thebase 1783 of SEQ ID NO. 80; 8) the base 1729 of SEQ ID NO. 81; 9) thebase 1781 of SEQ ID NO. 82; 10) the base 1774 of SEQ ID NO. 83; 11) thebase 1728 of SEQ ID NO. 84; and 12) the base 1644 of SEQ ID NO. 85.

Primer pairs used for the amplification reaction can be appropriatelyselected by those skilled in the art to preferably satisfy theconditions above on the basis of the base sequence of SEQ ID NO:27.Preferably, any primer pair having a base sequence selected from thegroup consisting of SEQ ID NOS: 39 and 40, SEQ ID NOS: 41 and 42, SEQ IDNOS: 43 and 44, SEQ ID NOS: 45 and 46, SEQ ID NOS: 47 and 48, SEQ IDNOS: 49 and 50, SEQ ID NOS: 51 and 52, and SEQ ID NOS: 53 and 54 isused. More preferably, the primer pair is selected from the groupconsisting of SEQ ID NOS: 45 and 46, and SEQ ID NOS: 47 and 48. Ifnecessary, the sequences of the above primer pairs containingsubstitutions, deletions or additions while retaining the bindingspecificity for the base sequence of the polymorphic site and adjacentregions thereto can also be used as primers.

To examine the resulting PCR product for restriction fragment lengthpolymorphisms, it is cleaved with restriction enzymes corresponding tothe restriction sites present in PCR markers. Such cleavage isaccomplished by incubation for several hours to a day at the recommendedreaction temperature for the restriction enzymes used. The PCR amplifiedsample cleaved with the restriction enzymes can be analyzed byelectrophoresis on an about 0.7%-2% agarose gel or an about 3% MetaPhor™agarose gel. The gel is visualized under UV light in ethidium bromide,for example.

In the most preferred embodiments of the present invention, restrictionenzyme cleavage patterns show the bands as shown in Table 2 below on thevisualized gel depending on the primer pair used.

TABLE 2 Approximate size (bp) of detected band Amplified with P4497 MobI(SEQ ID NOS: 39 and 40) Restriction enzyme MboI Test rice genome havingthe Rf-1 gene (homozygous): 730 no: 385, 345 Amplified with P9493 BslI(SEQ ID NOS: 41 and 42) Restriction enzyme BslI Test rice genome havingthe Rf-1 gene (homozygous): 126 no: 100, 26 Amplified with P23945 MboI(SEQ ID NOS: 43 and 44) Restriction enzyme MboI Test rice genome havingthe Rf-1 gene (homozygous): 160, 100 no: 260 Amplified with P41030 TaqI(SEQ ID NOS: 45 and 46) Restriction enzyme TaqI Test rice genome havingthe Rf-1 gene (homozygous): 280 no: 90, 190 Amplified with P45177 BstUI(SEQ ID NOS: 47 and 48) Restriction enzyme BstUI Test rice genome havingthe Rf-1 gene (homozygous): 20, 65, 730 no: 20, 65, 175, 555 Amplifiedwith B60304 MspI (SEQ ID NOS: 49 and 50) Restriction enzyme MspI Testrice genome having the Rf-1 gene (homozygous): 330 no: 220, 110Amplified with B59066 BsaJI (SEQ ID NOS: 51 and 52) Restriction enzymeBsaJI Test rice genome having the Rf-1 gene (homozygous): 420 no: 65,355 Amplified with B56691 XbaI (SEQ ID NOS: 53 and 54) Restrictionenzyme XbaI Test rice genome having the Rf-1 gene (homozygous): 670 no:140, 530

In Example 3 below, recombinants proximal to the Rf-1 gene having pollenfertility (RS1-RS2, RC1-RC8) were tested for the chromosomalorganization of the Rf-1 region using 14 polymorphic markers includingthe 8 primer pairs described above. As a result, it was confirmed thatall the plants carry the Rf-1 gene derived from the indica varietybetween P9493 BslI and 59066 BsaJI. This result showed that recombinantpollens having the chromosomal organization as shown in FIG. 3 havepollen fertility, i.e. the Rf-1 gene is functional in these pollens.This means that a sequence determining the presence of the function ofthe Rf-1 gene is included in the indica region common to theserecombinant pollens, i.e. in a region from the P4497 MboI to B56691 XbaIloci (about 65 kb) as estimated at maximum.

In the present invention, chromosomal walking was started on thepresumption that the S12564 Tsp509I locus should be vary proximal to theRf-1 locus as judged from the frequency of appearance of individuals bycrossing. In fact, the genetic distance between both loci has beencalculated to be about 0.04 cM as the result of the high-precisionsegregation analysis of the present invention. Even one of markers knownto be most closely linked to the Rf-1 locus as described in JapanesePatent Public Disclosure No. 2000-139465 is reported to have a geneticdistance of 1 cM from the Rf-1 locus. Considering that 1 cM is estimatedto be equivalent to 300 kb on average in rice, a considerable timeshould be required to restrict the Rf-1 gene region if chromosomalwalking were started from the marker described in Japanese Patent PublicDisclosure No. 2000-139465.

VI. Method for Inhibiting the Function of Rf-1 Gene to Restore Fertility

According to the present invention, the nucleic acid containing thelocus of a fertility restorer gene (Rf-1) including the nucleic acidswhich function to restore fertility was isolated. The entire basesequence thereof was determined, whereby the fertility restoringfunction of the Rf-1 gene can be controlled by genetic engineeringtechniques. Thus, the present invention further provides a method forinhibiting the function of Rf-1 to restore fertility.

A method for inhibiting the function of the Rf-1 gene to restorefertility according to one embodiment of the present invention comprisesintroducing an antisense having at least 100 continuous bases in length,and having a base sequence complementary to a nucleic acid having thebase sequence of SEQ ID NO. 27, or to a nucleic acid having a basesequence which is identical to at least 70% of the base sequence of SEQID NO. 27, and which functions to restore fertility.

In an embodiment, the method for inhibiting the function of the Rf-1gene to restore fertility according to the present invention comprisesintroducing an antisense having at least 100 continuous bases in length,and being selected from base sequences complementary to a nucleic acidhaving the base sequence of the bases 38538-54123, preferably the bases42357-53743, more preferably the bases 42132-48883 of SEQ ID NO:27, orto a nucleic acid having a base sequence which is identical to at least70% of the base sequence of the bases 38538-54123, preferably the bases42357-53743, more preferably the bases 42132-48883, still morepreferably the bases 42132-46318 of SEQ ID NO:27 and, which functions torestore fertility.

In an especially preferable embodiment, the method for inhibiting thefunction of the Rf-1 gene to restore fertility according to the presentinvention comprises introducing an antisense having at least 100 basesin length, and being selected from base sequences complementary to anucleic acid encoding the amino acid sequence of SEQ ID NO. 75, or anamino acid sequence which is identical to at least 70% of the amino acidsequence of SEQ ID NO. 75, and which functions to restore fertility.

Most preferably, the nucleic acid encoding the amino acid sequence ofSEQ ID NO. 75, or an amino acid sequence which is identical to at least70% of the amino acid sequence of SEQ ID NO. 75 is selected from nucleicacids of the following a)-p):

a) a nucleic acid comprising the bases 215-2587 of SEQ ID NO:69;

b) a nucleic acid comprising the bases 213-2585 of SEQ ID NO:70;

c) a nucleic acid comprising the bases 218-2590 of SEQ ID NO:71;

d) a nucleic acid comprising the bases 208-2580 of SEQ ID NO:72;

e) a nucleic acid comprising the bases 149-2521 of SEQ ID NO:73;

f) a nucleic acid comprising the bases 225-2597 of SEQ ID NO:74;

g) a nucleic acid comprising the bases 43907-46279 of SEQ ID NO:27;

h) a nucleic acid comprising the bases 229-2601 of SEQ ID NO:80;

i) a nucleic acid comprising the bases 175-2547 of SEQ ID NO:81;

j) a nucleic acid comprising the bases 227-2599 of SEQ ID NO:82;

k) a nucleic acid comprising the bases 220-2592 of SEQ ID NO:83;

l) a nucleic acid comprising the bases 174-2546 of SEQ ID NO:84;

m) a nucleic acid comprising the bases 90-2462 of SEQ ID NO:85;

n) a nucleic acid which is identical to at least 70% of the nucleic acidof any of a)-m), and which functions to restore fertility;

o) a nucleic acid which hybridizes to the nucleic acid of any of a)-m)under a moderate or high stringent condition, and which functions torestore fertility; and

p) a nucleic acid wherein one or a plurality of base(s) is deleted from,added to or substituted from the nucleic acid of any of a)-m), and whichfunctions to restore fertility.

The antisense has a length of at least 100 bases or more, morepreferably 500 bases or more, most preferably 1000 bases or more. Fromthe viewpoint of technical convenience of introduction, it preferablyhas a length of 10000 bases or less, more preferably 5000 bases or less.The antisense can be synthesized by known methods. The antisense can beintroduced into rice by known methods as described in e.g. Terada et al.(Plant Cell Physiol. 2000 July, 41(7), pp. 881-888).

It is also anticipated that Rf-1 disrupted lines can be established byscreening variant lines containing a transposable element such as, butnot limited to, Tos17 (Hirochika H. et al. 1996, Proc. Natl. Acad. Sci.USA 93, pp. 7783-7788) for a line containing the transposable element inthe base sequence of SEQ ID NO:27. In plants, gene disruption byhomologous recombination has been studied. It may also be possible toinhibit fertility restoring function by establishing such a line inwhich the Rf-1 gene has been replaced by a variant Rf-1 gene using anucleic acid having the base sequence of SEQ ID NO. 27, or a nucleicacid having a base sequence which is identical to at least 70% of thebase sequence of SEQ ID NO. 27.

REFERENCES

1. Fukuta et al. 1992, Jpn J. Breed. 42 (supl. 1) p. 164-165.

2. Japanese Patent Public Disclosure No. HEI7(1995)-222588.

3. Japanese Patent Public Disclosure No. HEI9(1997)-313187.

4. Japanese Patent Public Disclosure No. 2000-139465.

5. Harushima et al. 1998, Genetics 148 p. 479-494.

6. Michaels and Amasino 1998, The Plant Journal 14(3) p. 381-385.

7. Neff et al. 1998, The plant Journal 14(3) p. 387-392.

8. D. E. Harry, et al., Theor Appl Genet (1998) 97:p. 327-336.

9. Hiei et al., Plant Journal (1994), 6(2), p. 272-282.

10. Komari et al., Plant Journal (1996) 10, p. 165-174.

11. Ditto et al., Proc. Natl. Acad. Sci. USA (1980), 77: p. 7347-7351,

12. P. Vos et al., Nucleic Acids Res. Vol. 23, p. 4407-4414 (1995).

13. O. Parnaud, X. et al, Mol. Gen. Genet. (1996) 252:p. 597-607.

14. A. Konieczny et al., (1993), Plant J. 4(2) p. 403-410.

15. Edwards et al., Nucleic Acids Res. 8(6): 1349, 1991.

16. Murray M. G. et al., Nucleic Acids Res. 8(19):4321-5, 1980.

17. Terada et al., Plant Cell Physiol. 2000, July, 41(7), p. 881-888.

18. Hirochika H. et al. 1996, Proc. Natl. Acad. Sci. USA 93, p.7783-7788.

19. Cui, X., Wise, R. P. and Schanble, P. S. (1996) The rf2 nuclearrestorer gene of male-sterile T-cytoplasm maize. Science, 272, 1334-1336

20. Liu, F., Cui, X., Horner, H. T., Weiner, H. and Schnable, P. S.(2001) Mitochondrial aldehyde dehydrogenase activity is required formale fertility in maize. The Plant Cell, 13, 1063-1078

EXAMPLES

The following examples further illustrate the present invention but arenot intended to limit the technical scope of the invention. Thoseskilled in the art can readily add modifications/changes to the presentinvention on the basis of the description of the specification, andthose modifications/changes are included in the technical scope of thepresent invention.

Reference Examples

The following reference examples are based on the examples described inour prior application (Japanese Patent Application No. 2000-247204 filedAug. 17, 2000).

Reference Example 1 Conversion of RFLP Markers Around Rf-1 Gene to PCRMarkers

In this reference example, nine RFLP markers (i.e., R1877, G291, R2303,S12564, C1361, S10019, G4003, S10602 and G2155) around the locus of Rf-1gene were converted to PCR markers.

(1) Materials and Methods

The following nine RFLP markers, R1877, G291, R2303, S12564, C1361,S10019, G4003, S10602 and G2155, were purchased from the NationalInstitute of Agrobiological Sciences, the Ministry of Agriculture,Forestry and Fisheries of Japan. After determining the base sequences ofthe inserts in the vectors, experiments were conducted according to thefollowing procedures. Among rice varieties herein, Asominori belongs tojaponica, and IR24 belongs to indica.

(2) Preparation of Asominori Genomic Library

Total DNA was extracted from green leaves of Asominori by the CTABmethod. After partial digestion with MboI, the DNA was fractionatedaccording to size by NaCl density gradient centrifugation (6-20% lineargradient, 20° C., 37,000 rpm, 4 hr, total volume=12 mL). A portion ofeach fraction (about 0.5 mL) was subjected to electrophoresis andfractions containing 15-20 kb DNA were collected and purified. A librarywas constructed using Lambda DASH II (Stratagene) as a vector inaccordance with the attached protocol. Giga Pack III Gold (Stratagene)was used for packaging. After packaging, 500 μL of SM Buffer and 20 μLof chloroform were added. After centrifugation, 20 μL of chloroform wasadded to the supernatant to make a library solution.

XL-1 Blue MRA (P2) was infected with 5 μL of a 50-fold dilution of thelibrary solution, whereupon 83 plaques were formed. This corresponded to4.15×10⁵ pfu per library, and hence, it was calculated that the plaquescovered 8.3×10⁹ bp assuming that the average length of the insertedfragments was 20 kb. The library was therefore considered to have anadequate size for the rice genome (4×10⁸ bp).

(3) Isolation of Genomic Clones Containing R1877-, C1361- andG4003-Marker Regions.

As for C1361 and G4003, plasmids containing the RFLP marker probe wereisolated and subjected to restriction enzyme treatment andelectrophoresis to separate the RFLP marker probe portion; the desiredDNA was recovered on a DNA recovery filter (Takara SUPREC-01). As forR1877, primers were designed that were specific to both ends of themarker probe and PCR was performed with the total DNA of Asominori usedas a template; the amplification products were electrophoresed andrecovered by the method described above. The recovered DNA was labelledwith a Rediprime DNA Labelling System (Amersham Pharmacia) to prepare aprobe for screening the library. PCR was performed in the usual manner(this also applies to the following description).

Screening of the library was performed in the usual manner afterblotting the plaques onto Hybond-N+ (Amersham Pharmacia). After primaryscreening, areas of positive plaques were individually punched out,suspended in SM buffer and subjected to the second round of screening.After the second screening, the positive plaques were punched out andsubjected to the third round of screening to isolate a single plaque.

The isolated plaque of interest was suspended in SM buffer and primarymultiplication of the phage was performed by the plate lysate method.The resulting phage-enriched solution was subjected to secondarymultiplication by shake culture and the phage DNA was purified withLambda starter kit (QIAGEN).

For each marker, primary screening was conducted on eight plates. A 10μL aliquot of the library solution was employed per plate. After theprimary, second and third rounds of screening, four genomic clones inassociation with R1877 were isolated and three were isolated inassociation with each of C1361 and G4003.

(4) Conversion of R1877 to PCR Marker

The isolated genomic clones were analyzed to identify the causative siteof RFLP, or the EcoRI site that exists in IR24 (indica rice) but not inAsominori (japonica rice), thereby converting R1877 to a PCR marker.

Specifically, the four isolated clones were subjected to the followinganalyses. First, T3 and T7 primers were used to determine the basesequences at both ends of the insert in each clone. Then, primersextending outwardly from both ends of the marker probe were designed.They were combined with T3 and T7 primers to give a combination of fourprimers in total, and employed in PCR with each clone used as thetemplate.

In a separate step, each clone was digested with NotI and EcoRI, andelectrophoresed to estimate the insert size and the length of each EcoRIfragment.

These analyses revealed the relative positions of the individual clones.In RFLP analysis, marker probe R1877 was reported to detect an EcoRIfragment of 20 kb in Nipponbare (japonica rice) and one of 6.4 kb inKasalath (indica rice)(ftp://ftp.staff.or.jp/pub/geneticmap98/parentsouthern/chr10/R1877.JPG).This fact, taken together with the results of analysis described above,gave a putative position for the EcoRI site that existed in IR24 but notin Asominori. Hence, a primer combination (SEQ ID NO:1×SEQ ID NO:2) thatwas designed to amplify the nearby region was employed to performgenomic PCR over 30 cycles, each cycle consisting of 94° C.×1 min, 58°C.×1 min and 72° C.×2 min. The PCR product was treated with EcoRI andsubjected to electrophoresis on 0.7% agarose gel.

As a result, the expected polymorphisms were observed between Asominoriand IR24. By treatment with EcoRI, the PCR product (˜3200 bp) wascleaved to yield 1500 bp and 1700 bp fragments in IR24 but not inAsominori. Mapping of the marker was made with an RIL (recombinantinbred line) of Asominori-IR24 with the results that the PCR marker waslocated in the same region as that of RFLP marker locus R1877, therebyconfirming the conversion of RFLP marker R1877 to a PCR marker, whichwas named R1877 EcoRI in the present invention.

(5) Conversion of G4003 to PCR Marker

The isolated genomic clones were analyzed to identify the causative siteof RFLP, or the HindIII site that existed in Asominori but not in IR24,thereby converting G4003 to a PCR marker.

By performing analyses similar to those employed for R1877, the relativepositions of the three isolated clones were revealed. In RFLP analysis,marker probe G4003 was reported to detect a HindIII fragment of 3 kb inNipponbare (japonica rice) and one of 10 kb in Kasalath (indica rice)(ftp://ftp.staff.or.jp/pub/geneticmap98/parentsouthern/chr10/R1877.JPG).This report, taken together with the analyses described above, led to atemporary conclusion that the HindIII site that existed in Asominori butnot in IR24 would be at either one of two candidate sites. Hence, aprimer combination (SEQ ID NOS: 3 and 4) that was designed to amplifythe area in the neighborhood of each HindIII site was employed toperform genomic PCR over 35 cycles, each cycle consisting of 94° C.×30sec, 58° C.×30 sec and 72° C.×30 sec. The PCR product was treated withHindIII and subjected to electrophoresis on 2% agarose gel. As a result,the HindIII site within the marker probe was found to havepolymorphisms. By treatment with HindIII, the PCR product (362 bp) wascleaved to yield a 95 bp fragment and a 267 bp fragment in Asominori butnot in IR24. Mapping of the site demonstrated the conversion of RFLPmarker G4003 to a PCR marker, which was named G4003 HindIII (SEQ IDNO:19) in the present invention.

(6) Conversion of C1361 to PCR Marker

Primers were designed on the basis of the base sequence information ofthe isolated genomic clones. PCR was performed with the total DNAs ofAsominori and IR24 being used as a template and the PCR product wasrecovered by known methods after electrophoresis. Using the recoveredDNA as a template, the inventors analyzed the base sequence of each ofthe rice varieties with ABI Model 310 in search of mutations that wouldcause polymorphisms.

By performing analyses similar to those employed for R1877, approximaterelative positions of the three isolated clones could be established. Asit turned out, however, regions around the C1361 marker would bedifficult to amplify by PCR or determine their base sequences, andhence, it would not be easy to identify the causative site of RFLP.Hence, the inventors took notice of the region capable of yielding acomparatively long PCR product (2.7 kb) and made an attempt to create adCAPS marker.

Specifically, upon comparing the base sequences of the genomic PCRproducts of said region using Asominori and Koshihikari (both japonicarice) and Kasalath and IR24 (both indica rice), the inventors found sixsites of polymorphism between japonica and indica. One of these sixsites was used to create a dCAPS marker. To this end, with SEQ ID NO:5and SEQ ID NO:6 used as primers, PCR was performed over 35 cycles, eachcycle consisting of 94° C.×30 sec, 58° C.×30 sec and 72° C.×30 sec. ThePCR product was treated with MwoI and analyzed by electrophoresis on 3%MetaPhor™ agarose gel. In Asominori, cleavage occurred at two sites togive three observable bands of about 25 bp, 50 bp and 79 bp, but in IR24cleavage occurred at one site to give two observable bands of about 50bp and 107 bp. Mapping demonstrated the conversion of RFLP marker C1361to a PCR marker, which was named C1361 MwoI (SEQ ID NO:20) in thepresent invention.

(7) Conversion of G2155 to PCR Marker

Primers specific to both ends of the marker probe were designed and PCRwas performed with the total DNA of Asominori, Koshihikari, IR24 orIL216 (a line produced by introducing Rf-1 gene into Koshihikari by backcrossing; its genotype was Rf-1/Rf-1) being used as a template.Purification of the PCR product and searching for a mutation that wouldbe useful for providing restriction fragment polymorphisms wereperformed by the methods already described above.

Specifically, as a result of comparing the base sequences ofcorresponding regions of the varieties under test, mutations were foundat three sites between the variety/line (IR24 and IL216) having Rf-1gene and the variety (Asominori and Koshihikari) not having Rf-1 gene.One of the three sites was utilized to create a dCAPS marker. To thisend, SEQ ID NO:7 and SEQ ID NO:8 were used as primers to perform PCRover 35 cycles, each cycle consisting of 94° C.×30 sec, 58° C.×30 secand 72° C.×30 sec. The PCR product was treated with MwoI and analyzed byelectrophoresis on 3% MetaPhor™ agarose gel. In Asominori, cleavageoccurred at one site to give two observable bands of about 25 bp and 105bp, but in IR24, cleavage occurred at two sites to give three observablebands of about 25 bp, 27 bp and 78 bp. Mapping demonstrated theconversion of RFLP marker G2155 to a PCR marker, which was named G2155MwoI (SEQ ID NO:21) in the present invention.

(8) Conversion of G291 to PCR Marker

Primers specific to internal sequences of the marker probe were designedand used in various combinations to perform PCR to find a primercombination that could yield an amplification product of the expectedsize. Using the selected primer combination, the inventors performed PCRwith the total DNA of Asominori, Koshihikari, IR24 and IL216 used as atemplate. Purification of the PCR product and searching for a mutationthat could be utilized in providing restriction fragment polymorphismswere performed by the methods already described above.

Specifically, using the primers designed to be specific for the markerprobe sequence, the inventors performed genomic PCR of each varietyunder test and compared the base sequences of the products. As a result,mutations were found at four sites between the variety/line having Rf-1gene (IR24 and IL216) and the variety (Asominori and Koshihikari) nothaving Rf-1 gene. One of the four sites was used to create a dCAPSmarker. To this end, SEQ ID NO:9 and SEQ ID NO:10 were used as primersto perform PCR over 35 cycles, each cycle consisting of 94° C.×30 sec,58° C.×30 sec and 72° C.×30 sec. The PCR product was treated with MspIand analyzed by electrophoresis on 3% MetaPhor™ agarose gel. In thevarieties/lines having Rf-1 gene, cleavage occurred at two sites to givethree observable bands of about 25 bp, 49 bp and 55 bp, but in thevarieties not having Rf-1 gene, cleavage occurred at one site to givetwo observable bands of about 25 bp and 104 bp. Mapping demonstrated theconversion of RFLP marker G291 to a PCR marker, which was named G291MspI (SEQ ID NO:22) in the present invention.

(9) Conversion of R2303 to PCR Marker

Primers specific to internal sequences of the marker probe were designedand PCR was performed with the total DNA of Asominori (japonica rice)and IR24 and Kasalath (indica rice) used as a template. Purification ofthe PCR product and searching for a mutation that could be used forproviding restriction fragment polymorphisms were performed by themethods already described above.

As a result of comparing the base sequences of corresponding regions ofthe varieties under test, a mutation was found between japonica rice andindica rice. Since the mutation occurred at the BslI recognition site,the site was directly used to create a CAPS marker. To this end, SEQ IDNO:11 and SEQ ID NO:12 were used as primers and PCR was performed over30 cycles, each cycle consisting of 94° C.×1 min, 58° C.×1 min and 72°C.×2 min. The PCR product was treated with BslI and analyzed byelectrophoresis on 2% agarose gel. In japonica rice, cleavage occurredat one site to give two observable bands of about 238 bp and 1334 bp,but in indica rice, cleavage occurred at two sites to give threeobservable bands of about 238 bp, 655 bp and 679 bp. Mappingdemonstrated the conversion of RFLP marker R2303 to a PCR marker, whichwas named R2303 BslI (SEQ ID NO:23) in the present invention.

(10) Converting S10019 to PCR Marker

S10019 was converted to a PCR marker in accordance with the method (9)of converting R2303 to a PCR marker.

Specifically, as a result of comparing the base sequences ofcorresponding regions of the varieties under test, a mutation was foundbetween japonica rice and indica rice. Since the mutation occurred atthe BstUI recognition site, the site was directly used to create a CAPSmarker. To this end, SEQ ID NO:13 and SEQ ID NO:14 were used as primersand PCR was performed over 30 cycles, each cycle consisting of 94° C.×1min, 58° C.×1 min and 72° C.×1 min. The PCR product was treated withBstUI and analyzed by electrophoresis on 2% agarose gel. In japonicarice, cleavage occurred at one site to give two observable bands ofabout 130 bp and 462 bp, but in indica rice, cleavage occurred at twosites to give three observable bands of about 130 bp, 218 bp and 244 bp.Mapping demonstrated the conversion of RFLP marker S10019 to a PCRmarker, which was named S10019 BstUI (SEQ ID NO:24) in the presentinvention.

(11) Conversion of S10602 to PCR Marker

S10602 was converted to a PCR marker in accordance with the method (9)of converting R2303 to a PCR marker.

Specifically, as a result of comparing the base sequences ofcorresponding regions of the varieties under test, a mutation was foundbetween japonica rice and indica rice. The mutation was used to create aCAPS marker. To this end, SEQ ID NO:15 and SEQ ID NO:16 were used asprimers and PCR was performed over 33 cycles, each cycle consisting of94° C.×1 min, 58° C.×1 min and 72° C.×1 min. The PCR product was treatedwith KpnI and analyzed by electrophoresis on 2% agarose gel. In japonicarice, cleavage occurred at one site to give two observable bands ofabout 117 bp and 607 bp, but in indica rice, no cleavage occurred,giving only an observable band of 724 bp. Mapping demonstrated theconversion of RFLP marker S10602 to a PCR marker, which was named S10602KpnI (SEQ ID NO:25) in the present invention.

(12) Conversion of S12564 to PCR Marker

S12564 was converted to a PCR marker in accordance with the method ofconverting R2303 to a PCR marker.

Specifically, as a result of comparing the base sequences ofcorresponding regions of the varieties under test, a mutation was foundbetween japonica rice and indica rice. The mutation was used to create adCAPS marker. To this end, SEQ ID NO:17 and SEQ ID NO:18 were used asprimers and PCR was performed over 35 cycles, each cycle consisting of94° C.×30 sec, 58° C.×30 sec and 72° C.×30 sec. The PCR product wastreated with Tsp509I and analyzed by electrophoresis on 3% MetaPhor™agarose gel. In japonica rice, cleavage occurred at two sites to givethree observable bands of 26 bp, 41 bp and 91 bp, but in indica rice,cleavage occurred at one site to give two observable bands of 41bp and117 bp. Mapping demonstrated the conversion of RFLP marker S12564 to aPCR marker, which was named S12564 Tsp509I (SEQ ID NO:26) in the presentinvention.

Reference Example 2 Mapping of Rf-1 Gene Locus

DNA was extracted from 1042 seedlings of the F1 population produced bypollinating MS Koshihikari with MS-FR Koshihikari, and the DNA extractwas used in the analysis. MS Koshihikari (generation: BC10F1) wascreated by replacing the cytoplasm of Koshihikari with BT type malesterility cytoplasm. MS-FR Koshihikari was a line created by introducingRf-1 gene from IR8 (supplied from National Institute of AgrobiologicalSciences) into MS Koshihikari (the locus of Rf-1 gene beingheterozygous).

First, each individual was investigated for the genotype at two markerloci R1877 EcoRI and G2155 MwoI described in Reference example 1 thatwould presumably be located on opposite sides of the locus of Rf-1 gene.Japonica type homozygotes with respect to either locus R1877 EcRI orG2155 MwoI were regarded as recombinants between these two marker loci.Then, each of such recombinants was investigated for the genotypes ofG291 MspI, R2303 BslI, S12564 Tsp 509I, C1361 MwoI, S10019 BstUI, G4003HindIII and S10602 KpnI loci, and the positions of recombination wereidentified.

The genotype investigation with respect to R1877 EcoRI and G2155 MwoIloci revealed that 46 individuals were recombinants around the locus ofRf-1 gene. Genotypes of the marker loci around the locus of Rf-1 genewere investigated and the results are shown in Table 3.

TABLE 3 Genotypes of Marker Loci in Recombinant Individuals Around Rf-1Locus Locus 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23R1877 EcoRI J J J J J J J J H H H H H H H H H H H H H H H G291 MspI H JJ J J J J J H H H H H H H H H H H H H H H R2303 BslI H H J J J J J J H HH H H H H H H H H H H H H S12564 Tsp509I H H H H H H H J H H H H H H H HH H H H H H H C1361 MwoI H H H H H H H H J J H H H H H H H H H H H H HS10019 BstUI H H H H H H H H J J J J J J J J H H H H H H H G4003 HindIIIH H H H H H H H J J J J J J J J J J J J J J J S10602 KpnI H H H H H H HH J J J J J J J J J J J J J J J G2155 MwoI H H H H H H H H J J J J J J JJ J J J J J J J Locus 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 4041 42 43 44 45 46 R1877 EcoRI H H H H H H H H H H H H H H H H H H H H HH H G291 MspI H H H H H H H H H H H H H H H H H H H H H H H R2303 BslI HH H H H H H H H H H H H H H H H H H H H H H S12564 Tsp509I H H H H H H HH H H H H H H H H H H H H H H H C1361 MwoI H H H H H H H H H H H H H H HH H H H H H H H S10019 BstUI H H H H H H H H H H H H H H H H H H H H H HH G4003 HindIII J J J J J J J J J H H H H H H H H H H H H H H S10602KpnI J J J J J J J J J J J J J J J J J J J J J H H G2155 MwoI J J J J JJ J J J J J J J J J J J J J J J J J J: Homozygous Koshihikari type H:Heterozygous Koshihikari type/MS-FR Koshihikari type

As shown in Table 3, recombinant 8 homozygous for japonica at the S12564Tsp509I marker locus and recombinants 9 and 10 homozygous for japonicaat the C1361 Mwo marker locus were obtained. As all of theserecombinants restored fertility, the former was regarded as arecombinant between the Rf-1 and S12564 Tsp509I loci while the latterwere regarded as recombinants between the Rf-1 and C1361 MwoI loci,showing that the Rf-1 gene is located between the S12564 Tsp509I andC1361 MwoI loci. Based on the report that only pollens carrying the Rf-1gene have fertility in individuals having the BT type male sterilecytoplasm in the cross above (C. Shinjyo, JAPAN. J. GENETICS Vol. 44,No. 3:149-156 (1969)), the Rf-1 gene locus could be located on adetailed linkage map (FIG. 4).

Example 1 Acquisition of Recombinant Individuals Proximal to the Rf-1Locus

(Materials and Methods)

DNA was extracted from each of 4103 individuals of BC10F1 populationproduced by pollinating MS Koshihikari (generation: BC10F1) with MS-FRKoshihikari (generation: BC9F1, heterozygous at the Rf-1 locus), andgenotyped at the S12564 Tsp509I and C1361 MwoI loci in the same manneras described in Reference example 2 above. Individuals having a genotypehomozygous for Koshihikari at the S12564 Tsp509I locus were regarded asthose generated by recombination between the Rf-1 and S12564 Tsp509Iloci, while individuals having a genotype homozygous for Koshihikari atthe C1361 MwoI locus were regarded as those generated by recombinationbetween the Rf-1 and C1361 MwoI loci.

(Results and Discussion)

A survey of 4103 individuals revealed one recombinant individual betweenthe Rf-1 and S12564 Tsp509I loci and 6 recombinant individuals betweenthe Rf-1 and C1361 MwoI loci. The previous survey of 1042 individualsobtained by crossing in Reference example 2 above had already revealedone recombinant individual between the Rf-1 and S12564 Tsp509I loci and2 recombinant individuals between the Rf-1 and C1361 MwoI loci as shownin Table 3.

Thus, a total of 2 recombinant individuals between the Rf-1 and S12564Tsp509I loci and 8 recombinant individuals between the Rf-1 and C1361MwoI loci were able to be oabtained from 5145 individuals. These 10individuals were tested by high-precision segregation analysis in theexamples below.

Example 2 Chromosomal Walking

(1) First Chromosomal Walking

(Materials and Methods)

A genomic library was constructed from the genomic DNA of Asominorijaponica (not carrying Rf-1) using Lambda DASH II vector as described inReference example 1 and tested by chromosomal walking.

PCR was routinely performed using total DNA of Asominori as a templatein combination with the following primer pair:

5′-atcaggagccttcaaattgggaac-3′ (SEQ ID NO:29) and5′-ctcgcaaattgcttaattttgacc-3′ (SEQ ID NO:30)designed for a partial base sequence (Accession No. D47284) of RFLPprobe S12564. The resulting amplification products of about 1200 bp wereelectrophoresed on an agarose gel and then purified by QIAEXII (QIAGEN).The purified DNA was labeled with a rediprime DNA labelling system(Amersham Pharmacia) to give a library screening probe (probe A, FIG.1).

The library was routinely screened after plaques were blotted ontoHybond-N⁺ (Amersham Pharmacia). Single plaques were separated, afterwhich phage DNA was purified by the plate lysate method using LambdaMidi kit (QIAGEN).

(Results and Discussion)

The results of terminal base sequence analysis and restriction enzymefragment length analysis showed that two (WSA1 and WSA3) of 4 clonesobtained by screening were in a relative position as shown in FIG. 1.The Asominori genomic base sequences corresponding to WSA1 and WSA3 weredetermined by primer walking (DNA Sequencer 377, ABI).

(2) Second Chromosomal Walking

(Materials and Methods)

In addition to the Asominori genomic library described above, an IR24genomic library was similarly constructed from the genomic DNA of anindica variety IR24 (carrying Rf-1) and tested by chromosomal walking.

PCR was routinely performed using DNA of WSA3 as a template incombination with the following primer pair:

5′-tgaaggagttatgggtgcgtgacg-3′ (SEQ ID NO:31) and5′-ttgccgagcacacttgccatgtgc-3′ (SEQ ID NO:32)designed for the Asominori genomic base sequence determined in (1). Theresulting amplification products of 524 bp were purified and labeled bythe method described above to give a library screening probe (probe E,FIG. 1).

Library screening and phage DNA purification were performed by themethod described above.

(Results and Discussion)

The results of terminal base sequence analysis and restriction enzymefragment length analysis showed that one (WSE8) of 15 clones obtained byscreening of the Asominori genomic library was in a relative position asshown in FIG. 1. The Asominori genomic base sequence corresponding toWSE8 was determined by primer walking.

The results of terminal base sequence analysis and restriction enzymefragment length analysis showed that two (XSE1 and XSE7) of 7 clonesobtained by screening of the IR24 genomic library were in a relativeposition as shown in FIG. 1. The IR24 genomic base sequencescorresponding to XSE1 and XSE7 were determined by primer walking.

(3) Third Chromosomal Walking

(Materials and Methods)

The Asominori genomic library and IR24 genomic library described abovewere tested by chromosomal walking.

PCR was routinely performed using DNA of WSE8 as a template incombination with the following primer pair:

5′-gcgacgcaatggacatagtgctcc-3′ (SEQ ID NO:33) and5′-ttacctgccaagcaatatccatcg-3′ (SEQ ID NO:34)designed for the Asominori genomic base sequence determined in (2). Theresulting amplification products of 1159 bp were purified and labeled bythe method described above to give a library screening probe (probe F,FIG. 1).

Library screening and phage DNA purification were performed by themethod described above.

(Results and Discussion)

The results of terminal base sequence analysis and restriction enzymefragment length analysis showed that two (WSF5 and WSF7) of 8 clonesobtained by screening of the Asominori genomic library were in arelative position as shown in FIG. 1. The Asominori genomic basesequences corresponding to WSF5 and WSF7 were determined by primerwalking.

The results of terminal base sequence analysis and restriction enzymefragment length analysis showed that two (XSF4 and XSF20) of 13 clonesobtained by screening of the IR24 genomic library were in a relativeposition as shown in FIG. 1. The IR24 genomic base sequencescorresponding to XSF4 and XSF20 were determined by primer walking.

(4) Fourth Chromosomal Walking

(Materials and Methods)

The Asominori genomic library and IR24 genomic library described abovewere tested by chromosomal walking.

PCR was routinely performed using DNA of WSF7 as a template incombination with the following primer pair:

5′-aaggcatactcagtggagggcaag-3′ (SEQ ID NO:35) and5′-ttaacctgaccgcaagcacctgtc-3′ (SEQ ID NO:36)designed for the Asominori genomic base sequence determined in (3). Theresulting amplification products of 456 bp were purified and labeled bythe method described above to give a library screening probe (probe G,FIG. 1).

Library screening and phage DNA purification were performed by themethod described above.

(Results and Discussion)

The results of terminal base sequence analysis and restriction enzymefragment length analysis showed that two (WSG2 and WSG6) of 6 clonesobtained by screening of the Asominori genomic library were in arelative position as shown in FIG. 1. The Asominori genomic basesequences corresponding to WSG2 and WSG6 were determined by primerwalking.

The results of terminal base sequence analysis and restriction enzymefragment length analysis showed that three (XSG8, XSG16 and XSG22) of 14clones obtained by screening of the IR24 genomic library were in arelative position as shown in FIG. 1. The IR24 genomic base sequencescorresponding to XSG8, XSG16 and XSG22 were determined by primerwalking.

(5) Fifth Chromosomal Walking

(Materials and Methods)

The IR24 genomic library described above was tested by chromosomalwalking.

We perused the public website of TIGR (The Institute for GenomicResearch) and found that a BAC (Bacterial Artificial Chromosome) clone(Accession No. AC068923) containing RFLP marker S12564 had beendeposited with a public database (GenBank). This BAC clone contains thegenomic DNA of Nipponbare japonica and it was shown from base sequencecomparison to completely include the contig regions of Asominori andIR24 prepared in (1)-(4) (FIG. 2).

Thus, PCR was routinely performed using total DNA of IR24 as a templatein combination with the following primer pair:

5′-tggatggactatgtggggtcagtc-3′ (SEQ ID NO:37) and5′-agtggaagtggagagagtagggag-3′ (SEQ ID NO:38)designed to amplify a part of this BAC clone. The resultingamplification products of about 600 bp were purified and labeled by themethod described above to give a library screening probe (probe H, FIG.1).

Library screening and phage DNA purification were performed by themethod described above.

(Results and Discussion)

The results of terminal base sequence analysis and restriction enzymefragment length analysis showed that one (XSH18) of 15 clones obtainedby screening of the IR24 genomic library was in a relative position asshown in FIG. 1. The IR24 genomic base sequence corresponding to XSH18was determined by primer walking.

Example 3 High Precision Segregation Analysis

(1) Development of PCR Marker P4497 MboI

Comparison between the genomic base sequence corresponding to the IR24contig (SEQ ID NO:27) and the genomic base sequence corresponding to theAsominori contig (SEQ ID NO:28) determined in Example 2 revealed thatthe 1239th base of SEQ ID NO:27 is A while the 12631st base of SEQ IDNO:28 corresponding to said position is G.

For detecting this change, fragments of about 730 bp are first amplifiedby PCR from a region surrounding said position using the followingprimer pair:

P4497 MboI F:

5′-ccctccaacacataaatggttgag-3′ (SEQ ID NO:39)

-   -   (corresponding to bases 853-876 of SEQ ID NO:27)    -   (corresponding to bases 12247-12270 of SEQ ID NO:28) and        P4497 MboI R:

5′-tttctgccaggaaactgttagatg-3′ (SEQ ID NO:40)

-   -   (corresponding to bases 1583-1560 of SEQ ID NO:27)    -   (corresponding to bases 12975-12952 of SEQ ID NO:28).        The amplification products can be visualized by electrophoresis        on an agarose gel after treatment with MboI. Thus, the change        can be detected as a difference in mobility in the agarose gel        due to the difference in the length of DNA after MboI treatment        because the amplification products from Asominori DNA having an        MboI recognition sequence (GATC) are cleaved with MboI while the        amplification products from IR24 DNA are not cleaved with MboI        for the lack of the MboI recognition sequence.        (2) Development of PCR Marker P9493 BslI

Comparison between the genomic base sequence corresponding to the IR24contig (SEQ ID NO:27) and the genomic base sequence corresponding to theAsominori contig (SEQ ID NO:28) determined in Example 2 revealed thatthe 6227th base of SEQ ID NO:27 is A while the 17627th base of SEQ IDNO:28 corresponding to said position is C.

For detecting this change, fragments of 126 bp are first amplified byPCR from a region surrounding said position using the following primerpair:

P9493 BslI F:

5′-gcgatcttatacgcatactatgcg-3′ (SEQ ID NO:41)

-   -   (corresponding to bases 6129-6152 of SEQ ID NO:27)    -   (corresponding to bases 17529-17552 of SEQ ID NO:28) and        P9493 BslI R:

5′-aaagtctttgttccttcaccaagg-3′ (SEQ ID NO:42)

-   -   (corresponding to bases 6254-6231 of SEQ ID NO:27)    -   (corresponding to bases 17654-17631 of SEQ ID NO:28).        The amplification products can be visualized by electrophoresis        on an agarose gel after treatment with BslI. Thus, the change        can be detected as a difference in mobility in the agarose gel        due to the difference in the length of DNA after BslI treatment        because the amplification products from Asominori DNA having a        BslI recognition sequence (CCNNNNNNNGG) are cleaved with BslI        while the amplification products from IR24 DNA are not cleaved        with BslI for the lack of the BslI recognition sequence.

This marker was developed by applying the dCAPS method (Michaels andAmasino 1998, Neff et al., 1998). Specifically, g is substituted for aat the base 6236 of SEQ ID NO:27 and the base 17636 of SEQ ID NO:28 bythe use of P9493 BslI R primer described above. Thus, the fragments fromAsominori DNA come to have a sequence of CCtttccttGG at 17626-17636 ofSEQ ID NO:28 so that they are cleaved with BslI.

(3) Development of PCR Marker P23945 MboI

Comparison between the genomic base sequence corresponding to the IR24contig (SEQ ID NO:27) and the genomic base sequence corresponding to theAsominori contig (SEQ ID NO:28) determined in Example 2 revealed thatthe 20680th base of SEQ ID NO:27 is G while the 32079th base of SEQ IDNO:28 corresponding to said position is A.

For detecting this change, fragments of 260 bp are first amplified byPCR from a region surrounding said position using the following primerpair:

P23945 MboI F:

5′-gaggatttatcaaaacaggatggacg-3′ (SEQ ID NO:43)

-   -   (corresponding to bases 20519-20544 of SEQ ID NO:27)    -   (corresponding to bases 31918-31943 of SEQ ID NO:28) and        P23945 MboI R:

5′-tgggcggcagcagtggaggataga-3′ (SEQ ID NO:44)

-   -   (corresponding to bases 20778-20755 of SEQ ID NO:27)    -   (corresponding to bases 32177-32154 of SEQ ID NO:28).        The amplification products can be visualized by electrophoresis        on an agarose gel after treatment with MboI. Thus, the change        can be detected as a difference in mobility in the agarose gel        due to the difference in the length of DNA after MboI treatment        because the amplification products from IR24 DNA having an MboI        recognition sequence (GATC) are cleaved with MboI while the        amplification products from Asominori DNA are not cleaved with        MboI for the lack of the MboI recognition sequence.        (4) Development of PCR Marker P41030 TaqI

Comparison between the genomic base sequence corresponding to the IR24contig (SEQ ID NO:27) and the genomic base sequence corresponding to theAsominori contig (SEQ ID NO:28) determined in Example 2 revealed thatthe 45461st base of SEQ ID NO:27 is A while the 49164th base of SEQ IDNO:28 corresponding to said position is G.

For detecting this change, fragments of 280 bp are first amplified byPCR from a region surrounding said position using the following primerpair:

P41030 TaqI F:

5′-aagaagggagggttatagaatctg-3′ (SEQ ID NO:45)

-   -   (corresponding to bases 45369-45392 of SEQ ID NO:27)    -   (corresponding to bases 49072-49095 of SEQ ID NO:28) and        P41030 TaqI R:

5′-atatcaggactaacaccactgctc-3′ (SEQ ID NO:46)

-   -   (corresponding to bases 45648-45625 of SEQ ID NO:27)    -   (corresponding to bases 49351-49328 of SEQ ID NO:28).        The amplification products can be visualized by electrophoresis        on an agarose gel after treatment with TaqI. Thus, the change        can be detected as a difference in mobility in the agarose gel        due to the difference in the length of DNA after TaqI treatment        because the amplification products from Asominori DNA having a        TaqI recognition sequence (TCGA) are cleaved with TaqI while the        amplification products from IR24 DNA are not cleaved with TaqI        for the lack of the TaqI recognition sequence.        (5) Development of PCR Marker P45177 BstUI

Comparison between the genomic base sequence corresponding to the IR24contig (SEQ ID NO:27) and the genomic base sequence corresponding to theAsominori contig (SEQ ID NO:28) determined in Example 2 revealed thatthe 49609th base of SEQ ID NO:27 is A while the 53311st base of SEQ IDNO:28 corresponding to said position is G.

For detecting this change, fragments of 812 bp are first amplified byPCR from a region surrounding said position using the following primerpair:

P45177 BstUI F:

5′-acgagtagtagcgatcttccagcg-3′ (SEQ ID NO:47)

-   -   (corresponding to bases 49355-49378 of SEQ ID NO:27)    -   (corresponding to bases 53057-53080 of SEQ ID NO:28) and        P45177 BstUI R:

5′-cagcgtgaaactaaaaacggaggc-3′ (SEQ ID NO:48)

-   -   (corresponding to bases 50166-50143 of SEQ ID NO:27)    -   (corresponding to bases 53868-53845 of SEQ ID NO:28).        The amplification products can be visualized by electrophoresis        on an agarose gel after treatment with BstUI. Thus, the change        can be detected as a difference in mobility in the agarose gel        due to the difference in the length of DNA after BstUI treatment        because the amplification products from IR24 DNA having a BstUI        recognition sequence (CGCG) at two positions are cleaved into 3        fragments with BstUI while the amplification products from        Asominori DNA having the BstUI recognition sequence at three        positions are cleaved with BstUI into four fragments.        (6) Development of PCR Marker B60304 MspI

Comparison between the genomic base sequence corresponding to the IR24contig (SEQ ID NO:27) determined in Example 2 and the base sequence ofthe BAC clone described above (Accession No. AC068923) revealed that the56368th base of SEQ ID NO:27 is T while the base of AC068923corresponding to said position is C.

For detecting this change, fragments of about 330 bp are first amplifiedby PCR from a region surrounding said position using the followingprimer pair:

B60304 MspI F:

5′-atcccacatcatcataatccgacc-3′ (SEQ ID NO:49)

-   -   (corresponding to bases 56149-56172 of SEQ ID NO:27) and        B60304 MspI R:

5′-agcttctcccttggatacggtggcg-3′ (SEQ ID NO:50)

-   -   (corresponding to bases 56479-56455 of SEQ ID NO:27).        The amplification products can be visualized by electrophoresis        on an agarose gel after treatment with MspI. Thus, the change        can be detected as a difference in mobility in the agarose gel        due to the difference in the length of DNA after MspI treatment        because the amplification products from Nipponbare DNA having an        MspI recognition sequence (CCGG) are cleaved with MspI while the        amplification products from IR24 DNA are not cleaved with MspI        for the lack of the MspI recognition sequence.

This marker was developed by applying the dCAPS method. Specifically, tis substituted for g at base 56463 of SEQ ID NO:27 by the use of B60304MspI R primer. As a result, the MspI recognition sequence of bases56460-56463 of SEQ ID NO:27 changes from CCGG into ccgt so that thefragments from SEQ ID NO:27 become unable to be cleaved with MspI. Thus,the fragments from IR24 have no MspI recognition sequence, while DNAfrom Nipponbare has the MspI recognition sequence at one position in aregion corresponding to bases 56367-56370 of SEQ ID NO:27.

(7) Development of PCR Marker B59066 BsaJI

Comparison between the genomic base sequence corresponding to the IR24contig (SEQ ID NO:27) determined in Example 2 and the base sequence ofthe BAC clone described above (Accession No. AC068923) revealed that the57629th base of SEQ ID NO:27 is C while the base of AC068923corresponding to said position is CC.

For detecting this change, fragments of about 420 bp are first amplifiedby PCR from a region surrounding said position using the followingprimer pair:

B59066 BsaJI F:

5′-atttgttggttagttgcggctgag-3′ (SEQ ID NO:51)

-   -   (corresponding to bases 57563-57586 of SEQ ID NO:27) and        B59066 BsaJI R:

5′-gcccaaactcaaaaggagagaacc-3′ (SEQ ID NO:52)

-   -   (corresponding to bases 57983-57960 of SEQ ID NO:27).        The amplification products can be visualized by electrophoresis        on an agarose gel after treatment with BsaJI. Thus, the change        can be detected as a difference in mobility in the agarose gel        due to the difference in the length of DNA after BsaJI treatment        because the amplification products from Nipponbare DNA having a        BsaJI recognition sequence (CCNNGG) are cleaved with BsaJI while        the amplification products from IR24 DNA are not cleaved with        BsaJI for the lack of the BsaJI recognition sequence.        (8) Development of PCR Marker B56691 XbaI

Comparison between the genomic base sequence corresponding to the IR24contig (SEQ ID NO:27) determined in Example 2 and the base sequence ofthe BAC clone described above (Accession No. AC068923) revealed that the66267th base of SEQ ID NO:27 is G while the base of AC068923corresponding to said position is C.

For detecting this change, fragments of about 670 bp are first amplifiedby PCR from a region surrounding said position using the followingprimer pair:

B56691 XbaI F:

5′-cctcaagtctcccctaaagccact-3′ (SEQ ID NO:53)

-   -   (corresponding to bases 66129-66152 of SEQ ID NO:27) and        B56691 XbaI R:

5′-gctctactgctgataaaccgtgag-3′ (SEQ ID NO:54)

-   -   (corresponding to bases 66799-66776 of SEQ ID NO:27).        The amplification products can be visualized by electrophoresis        on an agarose gel after treatment with XbaI. Thus, the change        can be detected as a difference in mobility in the agarose gel        due to the difference in the length of DNA after XbaI treatment        because the amplification products from Nipponbare DNA having an        XbaI recognition sequence (TCTAGA) are cleaved with XbaI while        the amplification products from IR24 DNA are not cleaved with        XbaI for the lack of the XbaI recognition sequence.        (9) Development of PCR Marker B53627 BstZ17I

Comparison between the genomic base sequence corresponding to the IR24contig (SEQ ID NO:27) determined in Example 2 and the base sequence ofthe BAC clone described above (Accession No. AC068923) revealed that the69331st base of SEQ ID NO:27 is T while the base of AC068923corresponding to said position is C.

For detecting this change, fragments of about 620 bp are first amplifiedby PCR from a region surrounding said position using the followingprimer pair:

B53627 BstZ17I F:

5′-tggatggactatgtggggtcagtc-3′ (SEQ ID NO:55)

-   -   (corresponding to bases 68965-68988 of SEQ ID NO:27) and        B53627 BstZ17I R:

5′-agtggaagtggagagagtagggag-3′ (SEQ ID NO:56)

-   -   (corresponding to bases 69582-69559 of SEQ ID NO:27).        The amplification products can be visualized by electrophoresis        on an agarose gel after treatment with BstZ17I. Thus, the change        can be detected as a difference in mobility in the agarose gel        due to the difference in the length of DNA after BstZ17I        treatment because the amplification products from IR24 DNA        having a BstZ17I recognition sequence (GTATAC) are cleaved with        BstZ17I while the amplification products from Nipponbare DNA are        not cleaved with BstZ17I for the lack of the BstZ17I recognition        sequence.        (10) Development of PCR Marker B40936 MseI

Development of all the following PCR markers (10)-(12) relates to astudy of the base sequences corresponding to further downstream regions(3′) of base 76363 at the 3′ end of SEQ ID NO:27.

The following primer pair was designed for the base sequence of the BACclone described above (Accession No. AC068923):

5′-tacgacgccatttcactccattgc-3′ (SEQ ID NO:57) and5′-catttctctatgggcgttgctctg-3′. (SEQ ID NO:58)PCR was routinely performed using this primer pair in combination withtotal DNAs of MS-FR Koshihikari (genotype of the Rf-1 locus: Rf-1 Rf-1)and Koshihikari as templates. The resulting amplification products ofabout 1300 bp were electrophoresed on an agarose gel and then purifiedby QIAEXII (QIAGEN). Analysis of the base sequence of the purified DNAby a DNA sequencer 377 (ABI) showed several polymorphisms.

One of them can be detected by PCR amplification of a region surroundingsaid position using the following primer pair:

B40936 MseI F:

5′-acctgtaggtatggcaccttcaacac-3′ (SEQ ID NO:59)

-   -   and        B40936 MseI R:

5′-ccaaggaacgaagttcaaatgtatgg-3′. (SEQ ID NO:60)The amplification products can be visualized by electrophoresis on anagarose gel after treatment with MseI. Thus, the change can be detectedas a difference in mobility in the agarose gel due to the difference inthe length of DNA after MseI treatment because the amplificationproducts from MS-FR Koshihikari (Rf-1 Rf-1) DNA having an MseIrecognition sequence (TTAA) are cleaved with MseI while theamplification products from Koshihikari DNA are not cleaved with MseIfor the lack of the MseI recognition sequence.

This marker was developed by applying the dCAPS method.

(11) Development of PCR Marker B19839 MwoI

The following primer pair was designed for the base sequence of the BACclone described above (Accession No. AC068923):

5′-tgatgtgtttgggcatccctttcg-3′ (SEQ ID NO:61) and5′-gagataggggacgacagacacgac-3′. (SEQ ID NO:62)PCR was routinely performed using this primer pair in combination withtotal DNAs of MS-FR Koshihikari (genotype of the Rf-1 locus: Rf-1 Rf-1)and Koshihikari as templates. The resulting amplification products ofabout 1200 bp were electrophoresed on an agarose gel and then purifiedby QIAEXII (QIAGEN). Analysis of the base sequence of the purified DNAby a DNA sequencer 377 (ABI) showed several polymorphisms.

One of them can be detected by PCR amplification of a region surroundingsaid position using the following primer pair:

B19839 MwoI F:

5′-tcctatggctgtttagaaactgcaca-3′ (SEQ ID NO:63)

-   -   and        B19839 MwoI R:

5′-caagttcaaacataactggcgttg-3′. (SEQ ID NO:64)The amplification products can be visualized by electrophoresis on anagarose gel after treatment with MwoI. Thus, the change can be detectedas a difference in mobility in the agarose gel due to the difference inthe length of DNA after MwoI treatment because the amplificationproducts from Koshihikari DNA having an MwoI recognition sequence(GCNNNNNNNGC) are cleaved with MwoI while the amplification productsfrom MS-FR Koshihikari (Rf-1 Rf-1) DNA are not cleaved with MwoI for thelack of the MwoI recognition sequence.

This marker was developed by applying the dCAPS method.

(12) Development of PCR Marker B2387 BfaI

The following primer pair was designed for the base sequence of the BACclone described above (Accession No. AC068923):

5′-cactgtcctgtaagtgtgctgtgc-3′ (SEQ ID NO:65) and5′-caagcgtgtgataaaatgtgacgc-3′. (SEQ ID NO:66)PCR was routinely performed using this primer pair in combination withtotal DNAs of MS-FR Koshihikari (genotype of the Rf-1 locus: Rf-1 Rf-1)and Koshihikari as templates. The resulting amplification products ofabout 1300 bp were electrophoresed on an agarose gel and then purifiedby QIAEXII (QIAGEN). Analysis of the base sequence of the purified DNAby a DNA sequencer 377 (ABI) showed several polymorphisms.

One of them can be detected by PCR amplification of a region surroundingsaid position using the following primer pair:

B2387 BfaI F:

5′-tgcctactgccattactatgtgac-3′ (SEQ ID NO:67)

-   -   and        B2387 BfaI R:

5′-acatactaccgtaaatggtctctg-3′. (SEQ ID NO:68)The amplification products can be visualized by electrophoresis on anagarose gel after treatment with BfaI. Thus, the change can be detectedas a difference in mobility in the agarose gel due to the difference inthe length of DNA after BfaI treatment because the amplificationproducts from Koshihikari DNA having an BfaI recognition sequence (CTAG)are cleaved with BfaI while the amplification products from MS-FRKoshihikari (Rf-1 Rf-1) DNA are not cleaved with BfaI for the lack ofthe BfaI recognition sequence.(13) Segregation Analysis

Two recombinants between the Rf-1 and S12564 Tsp509I loci (RS1 and RS2)and 8 recombinants between the Rf-1 and C1361 MwoI loci (RC1 to RC8)obtained in Example 1 were genotyped at the 12 DNA marker loci developedin (1) to (12) above. The results are shown in Table 4 along with thegenotypes of each recombinant at the S12564 Tsp509I and C1361 MwoI loci.

TABLE 4 Genotypes of recombinants proximal to the Rf-1 locus at variousmarker loci Locus RS1 RS2 RC1 RC2 RC3 RC4 RC5 RC6 RC7 RC8 S12564 Tsp509IJ J H H H H H H H H P4497 MboI J J H H H H H H H H P9493 BslI H H H H HH H H H H P23945 MboI H H H H H H H H H H P41030 TaqI H H H H H H H H HH P45177 BstUI H H H H H H H H H H B60304 MspI H H H H H H H H H HB59066 BsaJI H H H H H H H H H H B56691 XbaI H H H H H H H J H H B53627BstZ17I H H H H H H H J H H B40936 MseI H H H H H H H J H H B19839 MwoIH H H H H J H J H H B2387 BfaI H H H H H J H J H J C1361 MwoI H H J J JJ J J J J J: Homozygous for Koshihikari H: Heterozygous forKoshihikari/MS-FR Koshihikari

Table 4 shows that all the recombinants have an indica-derived Rf-1chromosomal region between P9493 BslI and 59066 BsaJI. This resultshowed that recombinant pollens having the chromosomal organization asshown in FIG. 3 have pollen fertility, i.e. the Rf-1 gene is functionalin these pollens. This means that a sequence determining the presence ofthe function of the Rf-1 gene is included in the indica region common tothese recombinant pollens, i.e. in a region from the P4497 MboI toB56691 XbaI loci (about 65 kb) as estimated at maximum.

However, there is a possibility that it is important for the expressionof the genetic function of the Rf-1 gene that the Rf-1 gene is partiallyof the indica genotype, and that the genetic function may not besignificantly changed whether the remaining regions are of the japonicaor indica genotype. Therefore, it cannot be concluded that the commonindica region above (bases 1239-66267 of SEQ ID NO:27) completelycontains the entire Rf-1 gene. However, it is thought that at least SEQID NO:27 completely contains the entire Rf-1 gene for the followingreasons:

1) the size of a gene is normally several kilobases, and rarely exceeds10 kb;

2) the genomic base sequence of IR24 determined by the present invention(SEQ ID NO:27) completely contains the common indica region above;

3) the 5′ end of SEQ ID NO:27 is located 1238 bp upstream of the 5′ endof the common indica region above and forms a part of another gene(S12564); and

4) the 3′ end of SEQ ID NO:27 is located 10096 bp downstream of the 3′end of the common indica region above.

Example 4 Complementation Assay for a 9.7 kb Fragment from XSE1

(Materials and Methods)

The λ phage clone XSE1 (FIGS. 1 and 5) was completely digested with NotIand electrophoresed on an agarose gel. The separated 9.7 kb fragment(including bases 1-9657 of SEQ ID NO:27) was purified by QIAEXII(QIAGEN).

On the other hand, an intermediate vector pSB200 having ahygromycin-resistant gene cassette was prepared on the basis of pSB11(Komari et al., supra.). Specifically, a nopaline synthase terminator(Tnos) was first fused to a ubiquitin promoter and a ubiquitin intron(Pubi-ubiI). A hygromycin-resistant gene (HYG(R)) was inserted betweenubiI and Tnos of the resulting Pubi-ubiI-Tnos complex to give anassembly of Pubi-ubiI-HYG(R)-Tnos. This assembly was fused to aHindIII/EcoRI fragment of pSB11 to give pKY205. Linker sites for addingrestriction enzyme sites NotI, NspV, EcoRV, KpnI, SacI, EcoRI wereinserted into the Hind III site upstream of Pubi of this pKY205 to givepSB200 having a hygromycin-resistant gene cassette.

After the plasmid vector pSB200 was completely digested with NotI, DNAwas recovered by ethanol precipitation. The recovered DNA was dissolvedin TE solution and then dephosphorylated by CIAP (TAKARA). The reactionsolution was electrophoresed on an agarose gel, and then a vectorfragment was purified from the gel using QIAEXII (QIAGEN).

The two fragments prepared above, i.e. a 9.7 kb fragment from XSE1 and avector fragment were subjected to a ligation reaction using DNA LigationKit Ver. 1 (TAKARA). After the reaction, DNA was recovered by ethanolprecipitation. The recovered DNA was dissolved in pure water (preparedby a Millipore system) and then mixed with E. coli DH5a cells, and themixture was electroporated. After electroporation, the solution wascultured with shaking in LB medium (37° C., 1 hr) and then plated on anLB plate containing spectinomycin and warmed (37° C., 16 hr). Plasmidswere isolated from 24 of the resulting colonies. Their restrictionenzyme fragment length patterns and boundary base sequences wereanalyzed to select desired E. coli cells transformed with recombinantplasmids.

The E. coli cells selected above were used for triparental mating withthe Agrobacterium tumefaciens strain LBA4404/pSB1 (Komari et al., 1996)and the helper E. coli strain HB101/pRK2013 (Ditta et al., 1980)according to the method of Ditta et al. (1980). Plasmids were isolatedfrom 6 of the colonies formed on an AB plate containing spectinomycinand their restriction enzyme fragment length patterns were analyzed toselect desired Agrobacterium cells.

The Agrobacterium cells selected above were used to transform MSKoshihikari (having BT cytoplasm and a nucleus gene substantiallyidentical to Koshihikari) according to the method of Hiei et al. (1994).Necessary immature seeds of MS Koshihikari for transformation can beprepared by pollinating MS Koshihikari with Koshihikari.

Transformed plants were transferred to a greenhouse under long-dayconditions after acclimation. 48 individuals grown to a stage suitablefor transplantation were transplanted into 1/5000a Wagner pots (4individuals/pot), and transferred into a greenhouse under short-dayconditions 3-4 weeks after transplantation. About one month afterheading, seed fertility was tested on standing plants.

(Results and Discussion)

All of the 48 transformed individuals were sterile. This indicates thatthe 9.7 kb insert fragment does not contain at least the full-lengthRf-1 gene.

Example 5 Complementation Assay for a 14.7 kb Fragment from XSE7

(Materials and Methods)

The λ phage clone XSE7 (FIGS. 1 and 5) was completely digested withEcoRI and then DNA was recovered by ethanol precipitation. The recoveredDNA was dissolved in TE solution and then blunted by DNA Blunting Kit(TAKARA). The reaction solution was electrophoresed on an agarose gel toseparate a 14.7 kb fragment (including bases 2618-17261 of SEQ IDNO:27), which was purified by QIAEXII (QIAGEN).

On the other hand, the plasmid vector pSB200 was completely digestedwith SacI and then DNA was recovered by ethanol precipitation. Therecovered DNA was dissolved in TE solution and then dephosphorylated byCIAP (TAKARA) and DNA was recovered by ethanol precipitation. Therecovered DNA was dissolved in TE solution and then blunted by DNABlunting Kit (TAKARA). The reaction solution was electrophoresed on anagarose gel, and then a vector fragment was purified from the gel usingQIAEXII (QIAGEN).

The two fragments prepared above, i.e. the 14.7 kb fragment from XSE7and the vector fragment were subjected to a ligation reaction using DNALigation Kit Ver. 1 (TAKARA). Subsequently, transformed plants wereprepared and studied according to the method described in Example 4.

(Results and Discussion)

All of the 48 transformed individuals were sterile. This indicates thatthe 14.7 kb insert fragment does not contain at least the full-lengthRf-1 gene.

Example 6 Complementation Assay for a 21.3 kb Fragment from XSF4

(Materials and Methods)

The λ phage clone XSF4 (FIGS. 1 and 5) was partially digested with NotIand electrophoresed on an agarose gel. The separated 21.3 kb fragment(including bases 12478-33750 of SEQ ID NO:27) was purified by QIAEXII(QIAGEN).

On the other hand, the plasmid vector pSB200 was completely digestedwith NotI and then DNA was recovered by ethanol precipitation. Therecovered DNA was dissolved in TE solution and then dephosphorylated byCIAP (TAKARA). The reaction solution was electrophoresed on an agarosegel, and then a vector fragment was purified from the gel using QIAEXII(QIAGEN).

The two fragments prepared above, i.e. the 21.3 kb fragment from XSF4and the vector fragment were subjected to a ligation reaction using DNALigation Kit Ver. 1 (TAKARA). Subsequently, transformed plants wereprepared and studied according to the method described in Example 4.

(Results and Discussion)

All of the 48 transformed individuals were sterile. This indicates thatthe 21.3 kb insert fragment does not contain at least the full-lengthRf-1 gene.

Example 7 Complementation Assay for a 13.2 kb Fragment from XSF20

(Materials and Methods)

The λ phage clone XSF20 (FIGS. 1 and 5) was completely digested withSalI and then DNA was recovered by ethanol precipitation. The recoveredDNA was dissolved in TE solution and then blunted by DNA Blunting Kit(TAKARA). The reaction solution was electrophoresed on an agarose gel toseparate a 13.2 kb fragment (including bases 26809-40055 of SEQ IDNO:27), which was purified by QIAEXII (QIAGEN).

On the other hand, the plasmid vector pSB200 was completely digestedwith EcoRV and then DNA was recovered by ethanol precipitation. Therecovered DNA was dissolved in TE solution and then dephosphorylated byCIAP (TAKARA). The reaction solution was electrophoresed on an agarosegel, and then a vector fragment was purified from the gel using QIAEXII(QIAGEN).

The two fragments prepared above, i.e. the 13.2 kb fragment from XSF20and the vector fragment were subjected to a ligation reaction using DNALigation Kit Ver. 1 (TAKARA). Subsequently, transformed plants wereprepared and studied according to the method described in Example 4.

(Results and Discussion)

All of the 44 transformed individuals were sterile. This indicates thatthe 13.2 kb insert fragment does not contain at least the full-lengthRf-1 gene.

Example 8 Complementation Assay for a 16.2 kb Fragment from XSF18

(Materials and Methods)

The λ phage clone XSF18 is identical to XSF20 at the 5′ and 3′ ends(bases 20328 and 41921 of SEQ ID NO:27, respectively), but lacksinternal bases 33947-38591. Thus, it comprises bases 20328-33946 and38592-41921 of SEQ ID NO:27. This is because clone XSF18 was initiallyisolated but found to contain the above deletion during amplificationafter isolation, and therefore, the amplification step was freshly takento isolate a complete clone designated XSF20.

The λ phage clone XSF18 (FIG. 5) was completely digested with NotI andelectrophoresed on an agarose gel. The separated 16.2 kb fragment(including bases 21065-33946 and 38592-41921 of SEQ ID NO:27) waspurified by QIAEXII (QIAGEN).

On the other hand, the plasmid vector pSB200 was completely digestedwith NotI and then DNA was recovered by ethanol precipitation. Therecovered DNA was dissolved in TE solution and then dephosphorylated byCIAP (TAKARA). The reaction solution was electrophoresed on an agarosegel, and then a vector fragment was purified from the gel using QIAEXII(QIAGEN).

The two fragments prepared above, i.e. the 16.2 kb fragment from XSF18and the vector fragment were subjected to a ligation reaction using DNALigation Kit Ver. 1 (TAKARA). Subsequently, transformed plants wereprepared and studied according to the method described in Example 4.

(Results and Discussion)

All of the 48 transformed individuals were sterile (FIG. 6). Thisindicates that the 16.2 kb insert fragment does not contain at least thefull-length Rf-1 gene.

Example 9 Complementation Assay for a 12.6 kb Fragment from XSG22

(Materials and Methods)

The λ phage clone XSG22 (FIGS. 1 and 5) was partially digested with NotIand electrophoresed on an agarose gel. The separated 12.6 kb fragment(including bases 31684-44109 of SEQ ID NO:27) was purified by QIAEXII(QIAGEN).

On the other hand, the plasmid vector pSB200 was completely digestedwith NotI and then DNA was recovered by ethanol precipitation. Therecovered DNA was dissolved in TE solution and then dephosphorylated byCIAP (TAKARA). The reaction solution was electrophoresed on an agarosegel, and then a vector fragment was purified from the gel using QIAEXII(QIAGEN).

The two fragments prepared above, i.e. the 12.6 kb fragment from XSG22and the vector fragment were subjected to a ligation reaction using DNALigation Kit Ver. 1 (TAKARA). Subsequently, transformed plants wereprepared and studied according to the method described in Example 4.

(Results and Discussion)

All of the 48 transformed individuals were sterile. This indicates thatthe 12.6 kb insert fragment does not contain at least the full-lengthRf-1 gene.

Example 10 (1) Complementation Assay for a 15.7 kb Fragment from XSG16

(Materials and Methods)

The λ phage clone XSG16 (FIGS. 1 and 5) was partially digested with NotIand electrophoresed on an agarose gel. The separated 15.7 kb fragment(including bases 38538-54123 of SEQ ID NO:27) was purified by QIAEXII(QIAGEN).

On the other hand, the plasmid vector pSB200 was completely digestedwith NotI and then DNA was recovered by ethanol precipitation. Therecovered DNA was dissolved in TE solution and then dephosphorylated byCIAP (TAKARA). The reaction solution was electrophoresed on an agarosegel, and then a vector fragment was purified from the gel using QIAEXII(QIAGEN).

The two fragments prepared above, i.e. the 15.7 kb fragment from XSG16and the vector fragment were subjected to a ligation reaction using DNALigation Kit Ver. 1 (TAKARA). Subsequently, transformed plants wereprepared and studied according to the method described in Example 4.

(Results and Discussion)

Of the 47 transformed individuals, at least 37 individuals clearlyrestored fertility (FIG. 6). This indicates that 15586 bases (bases38538-54123 of SEQ ID NO:27) derived from rice (IR24) in the 15.7 kbinsert fragment include the full-length Rf-1 gene.

(2) Complementation Assay for an Internal 11.4 kb Fragment in XSG16

(Materials and Methods)

The λ phage clone XSG16 was completely digested with AlwNI and BsiWI andthen DNA was recovered by ethanol precipitation. The recovered DNA wasdissolved in TE solution and then blunted by DNA Blunting Kit (TAKARA).The reaction solution was electrophoresed on an agarose gel to separatea 11.4 kb fragment, which was purified by QIAEXII (QIAGEN).

The plasmid vector pSB11 (Komari et al. Plant Journal, 1996) wascompletely digested with SmaI and then DNA was recovered by ethanolprecipitation. The recovered DNA was dissolved in TE solution and thendephosphorylated by CIAP (TAKARA). The reaction solution waselectrophoresed on an agarose gel, and then a vector fragment waspurified from the gel using QIAEXII (QIAGEN).

The two fragments prepared above were subjected to a ligation reactionusing DNA Ligation Kit Ver. 1 (TAKARA). After the reaction, DNA wasrecovered by ethanol precipitation. The recovered DNA was dissolved inpure water (prepared by a Millipore system) and then mixed with E. coliDH5a cells, and the mixture was electroporated. After electroporation,the solution was cultured with shaking in LB medium (37° C., 1 hr) andthen plated on an LB plate containing spectinomycin and warmed (37° C.,16 hr). Plasmids were isolated from 14 of the resulting colonies, andtheir restriction enzyme fragment length patterns and boundary basesequences were analyzed to select desired E. coli cells.

The E. coli cells selected above were used for triparental mating withthe Agrobacterium tumefaciens strain LBA4404/pSB4U (Takakura et al.,Japanese Patent Application No. 2001-269982 (WO02/019803 A1)) and thehelper E. coli strain HB101/pRK2013 (Ditta et al., 1980) according tothe method of Ditta et al. (1980). Plasmids were isolated from 12 of thecolonies formed on an AB plate containing spectinomycin and theirrestriction enzyme fragment length patterns were analyzed to selectdesired Agrobacterium cells.

The Agrobacterium cells selected above were used to transform MSKoshihikari (having BT cytoplasm and a nucleus gene substantiallyidentical to Koshihikari) according to the method of Hiei et al. (1994).Necessary immature seeds of MS Koshihikari for transformation can beprepared by pollinating MS Koshihikari with Koshihikari.

Transformed plants were transferred to a greenhouse under long-dayconditions after acclimation. 120 individuals grown to a stage suitablefor transplantation were transplanted into 1/5000a Wagner pots (4individuals/pot), and transferred into a greenhouse under short-dayconditions about one month after transplantation. About one month afterheading, one typical ear was sampled from each plant to evaluate seedfertility (the percentage of fertile paddies to total paddies).

(Results and Discussion)

Of the 120 transformed individuals, 59 individuals showed seed fertilityof 10% or more, among which 19 individuals showed seed fertility of 70%or more. This indicates that the 11.4 kb insert fragment (bases42357-53743 of SEQ ID NO:27) contains an essential Rf-1 gene region forexpressing a fertility restoring function.

(3) Complementation Assay for an Internal 6.8 kb Fragment in XSG16

(Materials and Methods)

The λ phage clone XSG16 was completely digested with HpaI and AlwNI andelectrophoresed on an agarose gel. The separated 6.8 kb fragment waspurified by QIAEXII (QIAGEN).

The subsequent procedures including the preparation of the plasmidvector pSB11 were performed according to the method in (2) above.

(Results and Discussion)

Of the 120 transformed individuals, 67 individuals showed seed fertilityof 10% or more, among which 26 individuals showed seed fertility of 70%or more. This indicates that the 6.8 kb insert fragment (bases42132-48883 of SEQ ID NO:27) contains an essential Rf-1 gene region forexpressing a fertility restoring function.

Example 11 Complementation Assay for a 16.9 kb Fragment from XSG8

(Materials and Methods)

The λ phage clone XSG8 (FIGS. 1 and 5) was completely digested with NotIand electrophoresed on an agarose gel. The separated 16.9 kb fragment(including bases 46558-63364 of SEQ ID NO:27) was purified by QIAEXII(QIAGEN).

On the other hand, the plasmid vector pSB200 was completely digestedwith NotI and then DNA was recovered by ethanol precipitation. Therecovered DNA was dissolved in TE solution and then dephosphorylated byCIAP (TAKARA). The reaction solution was electrophoresed on an agarosegel, and then a vector fragment was purified from the gel using QIAEXII(QIAGEN).

The two fragments prepared above, i.e. the 16.9 kb fragment from XSG8and the vector fragment were subjected to a ligation reaction using DNALigation Kit Ver. 1 (TAKARA). Subsequently, transformed individuals wereprepared and studied according to the method described in Example 4.

(Results and Discussion)

All of the 48 transformed individuals were sterile. This indicates thatthe 16.9 kb insert fragment does not contain at least the full-lengthRf-1 gene.

Example 12 Complementation Assay for a 20.0 kb Fragment from XSH18

(Materials and Methods)

The λ phage clone XSH18 (FIGS. 1 and 5) was completely digested withNotI and electrophoresed on an agarose gel. The separated 20.0 kbfragment (including bases 56409-76363 of SEQ ID NO:27) was purified byQIAEXII (QIAGEN).

On the other hand, the plasmid vector pSB200 was completely digestedwith NotI and then DNA was recovered by ethanol precipitation. Therecovered DNA was dissolved in TE solution and then dephosphorylated byCIAP (TAKARA). The reaction solution was electrophoresed on an agarosegel, and then a vector fragment was purified from the gel using QIAEXII(QIAGEN).

The two fragments prepared above, i.e. the 20.0 kb fragment from XSH18and the vector fragment were subjected to a ligation reaction using DNALigation Kit Ver. 1 (TAKARA). Subsequently, transformed individuals wereprepared and studied according to the method described in Example 4.

(Results and Discussion)

All of the 44 transformed individuals were sterile. This indicates thatthe 20.0 kb insert fragment does not contain at least the full-lengthRf-1 gene.

Example 13 Complementation Assay for a 19.7 kb Fragment from anOverlapping Region of XSG8 and XSH18

(Materials and Methods)

A plasmid (XSG8SB200F) isolated from desired E. coli cells obtained byligation in Example 11 was completely digested with SalI and StuI andelectrophoresed on an agarose gel. The separated 12.8 kb fragment(including bases 50430-63197 of SEQ ID NO:27) was purified by QIAEXII(QIAGEN).

On the other hand, a plasmid (XSH18SB200R) isolated from desired E. colicells obtained by ligation in Example 12 was completely digested withSalI, StuI and XhoI and electrophoresed on an agarose gel to separate a6.9 kb fragment (including bases 63194-70116 of SEQ ID NO:27), which waspurified by QIAEXII (QIAGEN).

Further, the plasmid vector pSB200 was completely digested with EcoRVand then DNA was recovered by ethanol precipitation. The recovered DNAwas dissolved in TE solution and then dephosphorylated by CIAP (TAKARA).The reaction solution was electrophoresed on an agarose gel, and then avector fragment was purified from the gel using QIAEXII (QIAGEN).

The three fragments prepared above, i.e. the 12.8 kb fragment from XSG8,the 6.9 kb fragment from XSH18 and the vector fragment were subjected toa ligation reaction using DNA Ligation Kit Ver. 1 (TAKARA). The ligationproduct contains a 19.7 kb fragment from an overlapping region of XSG8and XSH18 (including 50430-70116 of SEQ ID NO:27) (XSX1 in FIG. 5).Subsequently, transformed individuals were prepared and studiedaccording to the method described in Example 4.

(Results and Discussion)

All of the 40 transformed individuals were sterile. This indicates thatthe 19.7 kb insert fragment does not contain at least the full-lengthRf-1 gene.

Example 14 Preparation of cDNA Library

Firstly, IL216, a line wherein the Rf-1 is introduced into Koshihikarivia backcrossing (the genotype, Rf-1/Rf-1), was prepared. The IL216 wasgrown in a greenhouse by a conventional method, and young panicles weresampled during the growth stage wherein the length between auricles is−5˜5 cm. Total RNA was extracted by the SDS-phenol method (Watanabe, A.and Price, C. A. (1982) Translation of mRNAs for subunits of chloroplastcoupling factor 1 in spinach. Proceedings of the National Academy ofSciences of the U.S.A., 79, 6304-6308), and the poly (A)⁺ RNA waspurified using QuickPrep mRNA Purification Kit (Amersham PharmaciaBiotech).

The purified poly (A)+ RNA was provided to prepare a cDNA library byZAP-cDNA Synthesis Kit (Stratagene). The titer of the prepared library(1 ml) was calculated to be 16,000,000 pfu/ml, and was determined to besufficiently large.

Example 15 Screening of the cDNA Library

(1) Preparation of the Screening Primers

PCR was performed by using the following two types of primes:

Sense primer 5′-tctcattctctccacgccctgctc-3′ (SEQ ID NO:76) Antisenseprimer 5′-acggcggagcaattcgtcgaacac-3′ (SEQ ID NO:77)and XSG16, a genomic clone of IR24, as a template. SEQ ID NOS:76 and 77correspond to the bases 43733-43756 and the bases 44038-44015 of SEQ IDNO:27, respectively.

After the electrophoresis, the amplification product of about 300 bp wasrecovered from the agarose gel by QIAEX II Gel Extraction Kit (QIAGEN).The recovered fragment was ³²P-labeld by Rediprime II DNA labellingsystem (Amersham Pharmacia Biotech) (The fragment is hereunder referredto as “Probe P”).

Further, PCR was performed by using the following two types of primes:

Sense primer 5′-agtgtgtggcatggtgcatttccg-3′ (SEQ ID NO:78) Antisenseprimer 5′-ctctacaggatacacggtgtaagg-3′ (SEQ ID NO:79)and XSG16, a genomic clone of IR24, as a template. SEQ ID NOS:78 and 79correspond to the bases 48306-48329 and the bases 50226-50203 of SEQ IDNO:27, respectively. After the electrophoresis, the amplificationproduct of about 1900 bp was recovered from the agarose gel. Therecovered fragment was ³²P-labeld by the method mentioned above (Thefragment is hereunder refers to as “Probe Q”).(2) Screening of the cDNA Libbary

The cDNA library prepared in Example 14 was provided to prepare 70 ofagar medium wherein about 15000 plaques appeared. Plaque lift wasperformed twice for each agar medium, and the plaques were transferredto Hybond-N⁺ (Amersham Pharmacia Biotech). One membrane was used forhybridization with Probe P, and the other membrane was used forhybridization with Probe Q. The whole steps were performed according tothe manufacture's instructions.

Probes were added to a hybridization solution containing 250M Na₂HPO₄, 1mM EDTA and 7% SDS, and hybridization was performed at 65° C. for 16hours. Washing was performed twice with a solution containing 1×SSC and0.1% SDS, at 65° C. for 15 minutes, and then twice with a solutioncontaining 0.1×SSC and 0.1% SDS, at 65° C. for 15 minutes. After thewashing, the membranes were analyzed with FUJIX BAS 1000 (Fuji PhotoFilms).

As a result, 8 plaques which showed positive for both Probe P and ProbeQ were identified. Therefore, those plaques were isolated, subclonedinto pBluescript according to the instructions of the manufacture(Stratagene). Among 8 clones, the terminal base sequences of 6 cloneswere identical to that of XSG16. The entire base sequences of the 6clones were determined, and the results are shown in SEQ ID NOS:69-74 inthe sequence listing.

All of the sequences, SEQ ID NOS:69-74 are presumed to encode a proteinhaving the amino acids 1-791 of SEQ ID NO:75. Specifically, all and eachof the 215-2587 of SEQ ID NO:69, the bases 213-2585 of SEQ ID NO:70, thebases 218-2590 of SEQ ID NO:71, the bases 208-2580 of SEQ ID NO:72, thebases 149-2521 of SEQ ID NO:73 and the bases 225-2597 of SEQ ID NO:74encodes a protein having amino acids 1-791 of SEQ ID NO:75. The abovebase sequences correspond to the bases 43907-46279 of SEQ ID NO:27.

The amino acid sequence of SEQ ID NO:75 was compared with the presumedamino acid sequence of the corn fertility restorer gene (Rf2), and theN-terminal 7 amino acid residues (Met-Ala-Arg-Arg-Ala-Ala-Ser) in bothamino acid sequences were concurred. These 7 amino acid residues areconsidered to be a portion of a targeting signal to mitochondria (Liu etal., 2001). Based on the above facts, the cDNAs isolated on thisoccasion are considered to contain the full coding region of the Rf-1gene. No homology between the amino acid sequences of the rice Rf-1 andthe corn Rf2 can be found except for the above region. It is presumedthat the mechanisms by which the gene products of the Rf-1 and the Rf2can restore fertility after being transferred to mitochondria aredistinct from each other.

In addition, the sequences of cDNAs isolated on this occasion werecompared with the genome sequence of IR24 (SEQ ID NO:27), and thestructures of exons and introns of the Rf-1 gene were clarified (FIG.7). As a result, it was shown that various transcription productswherein the splicing forms and the poly A addition positions aredifferent, are present in a plant body. There is no intron in the codingregion of the Rf-1 gene.

Example 16 Complementation Assay

A complementation assay was performed by using a 4.2 kb fragmentcontaining the promoter region and the presumed translation region ofthe Rf-1 gene. The 4.2 kb fragment is in a plasmid containing the 6.8 kbgenome derive from IR24 which proved to have fertility restorer functionin Example 10(3).

Firstly, the plasmid described in Example 10(3) was treated with EcoRI,and was subjected to electrophoresis with agarose gel. The 4.2 kbfragment containing the promoter region and the presumed translationregion of the Rf-1 (corresponding to the bases 42132-46318 in SEQ IDNO:27) was separated, recovered from the gal using QIAEXII (QIAGEN). The4.2 kb fragment was subjected to ligation reaction using DNA LigationKit Ver. 1 (TAKARA) together with pBluescript II SK (−) which has beentreated with EcoRI and then with CIAP (TAKARA). After the reaction, theDNA was recovered by ethanol precipitation.

The recovered DNA was dissolved in pure water (prepared by a Milliporesystem) and then mixed with E. coli DH5a cells, and the mixture waselectroporated. After electroporation, the solution was cultured byshaking in LB medium (37° C., 1 hr) and then plated on an LB platecontaining ampicillin and warmed (37° C., 16 hr). Plasmids were isolatedfrom 12 of the resulting colonies, and their restriction enzyme fragmentlength patterns and boundary base sequences were analyzed to selectdesired E. coli cells. Then, plasmids isolated from the selected E. coliwere treated with BamHI and SalI, and electrophoresed on an agarose gel.The 4.2 kb fragment containing the promoter region and the presumedtranslation region of Rf-1 was separated, and recovered from the gelusing QIAEXII (QIAGEN).

On the other hand, TnosJH0072 (an intermediate vector comprising the nosterminator and a cassette of the ampicillin resistant gene) was treatedwith BamHI and SalI, and electrophored on a agarose gel. The 3.0 kbfragment containing the nos terminator and the ampicillin-resistant genewas separated, and was recovered from the gel using QIAEXII (QIAGEN).

The 4.2 kb fragment containing the promoter region and the presumedtranslation region of Rf-1, and the fragment derived from TnosJH0072were subjected to ligation reaction, and to electroporation by themethods discussed above. The reactant was spread on LB plates containingampicillin, and incubated (37° C., 16 hr). Plasmids were isolated from12 of the resulting colonies, and their restriction enzyme fragmentlength patterns and boundary base sequences were analyzed to selectdesired E. coli cells.

Further, plasmids isolated from the selected E. coli were treated withSgfI, and electrophoresed on an agarose gel. The 4.2 kb fragmentcontaining the promoter region and the presumed translation region ofRf-1 was separated, and recovered from the gel using QIAEXII (QIAGEN).The 4.2 kb fragment and pSB200Pac (an intermediate vector comprising acassette of the hygromycin-resistant gene) which has been treated withPacI and then with CIAP (TAKARA) were subjected to ligation reaction,and to electroporation by the methods discussed above. The reactant wasspread on LB plates containing spectinomycin, and incubated (37° C., 16hr). Plasmids were isolated from 16 of the resulting colonies, and theirrestriction enzyme fragment length patterns and boundary base sequenceswere analyzed to select desired E. coli cells.

As a result of the above steps, E. coli cells were obtained wherein thechimera gene of the fragment containing the promoter region of the Rf-1and the presumed translation region of the Rf-1 attached with the nosterminator has been inserted within an intermediate vector. The E. colicells were used for triparental mating with the Agrobacteriumtumefaciens strain LBA4404/pSB1 (Komari et al., 1996) and the helper E.coli strain HB101/pRK2013 (Ditta et al., 1980) according to the methodof Ditta et al. (1980). Plasmids were isolated from 6 of the coloniesformed on an AB plate containing spectinomycin and their restrictionenzyme fragment length patterns were analyzed to select desiredAgrobacterium cells.

The Agrobacterium cells selected above were used to transform MSKoshihikari (having BT cytoplasm and a nucleus gene substantiallyidentical to Koshihikari) according to the method of Hiei et al. (1994).Necessary immature seeds of MS Koshihikari for transformation wereprepared by pollinating MS Koshihikari with Koshihikari.

Transformed plants were transferred to a greenhouse under long-dayconditions after acclimation. 32 individuals grown to a stage suitablefor transplantation were transplanted into 1/5000a Wagner pots (4individuals/pot), and transferred into a greenhouse under short-dayconditions 3-4 weeks after transplantation. About one month afterheading, seed fertility was tested on standing plants. As a result, 28individuals among the 32 transformed individuals restored fertility.

By the above procedures, it has been experimentally demonstrated thatthe function of the Rf-1 gene can be furnished by expressing thepresumed translation region.

Example 17 Isolation of cDNA

In Example 15, the cDNA library derived from IL216 young panicles wasscreened with Probe P and Probe Q. Plaques which are positive for bothprobes were isolated and analyzed, and 6 cDNA were isolated. In thisexample, similar screening was performed with Probe P and Probe R asmentioned below, and six additional cDNAs were isolated. Details are asfollows.

Firstly, PCR was performed by using the following two types of primes:

Sense primer 5′-cagttgggttgaaacctaatactg-3′ (SEQ ID NO:86) Antisenseprimer 5′-cactaaaccgttagacgagaaagc-3′ (SEQ ID NO:87)and a genomic clone of IR24, XSG16 as a template. SEQ ID NOS:86 and 87correspond to the bases 45522-45545 and the bases 45955-45932 of SEQ IDNO:27, respectively.

After the electrophoresis, the amplification product of about 430 bp wasrecovered from the agarose gel by QIAEX II (QIAGEN). The recoveredfragment was ³²P-labeld by Rediprime II DNA labelling system (AmershamPharmacia Biotech) (hereinafter referred as “Probe R”, FIG. 8).

The cDNA library derived from IL216 young panicles was provided toprepare 20 of agar medium wherein about 15000 plaques appeared. Plaquelift was performed twice for each agar medium, and the plaques weretransferred to Hybond-N⁺ (Amersham Pharmacia Biotech). One membrane wasused for hybridization with Probe P of Example 15, and the othermembrane was used for hybridization with Probe R. All of the steps wereperformed according to the manufacture's instructions. As a result, 12plaques were identified which proved to be positive for both Probe P andProbe R.

Accordingly, those plaques were isolated, and subcloned into pBluescriptaccording to the instructions of the manufacture (Staratagene). Theterminal base sequences of the cones were determined. Among 12 clones,the terminal base sequences of 6 clones were identical to that of XSG16,and thus the entire base sequences of those 6 clones were determined(#7-#12). The results were shown in SEQ ID NOS:80-85.

All of the sequences, SEQ ID NOS:80-85 are presumed to encode a proteinhaving the amino acids 1-791 of SEQ ID NO:75. Specifically, all and eachof the 229-2601 of SEQ ID NO:80, the bases 175-2547 of SEQ ID NO:81, thebases 227-2599 of SEQ ID NO:82, the bases 220-2592 of SEQ ID NO:83, thebases 174-2546 of SEQ ID NO:84 and the bases 90-2462 of SEQ ID NO:85encodes a protein having amino acids 1-791 of SEQ ID NO:75. The abovebase sequences correspond to the bases 43907-46279 of SEQ ID NO:27.

The sequences of cDNAs isolated on this occasion were compared with thegenome sequence of IR24 (SEQ ID NO:27), and the structures of exons andintrons were clarified (FIG. 8). Among the cDNAs isolated on thisoccasion, there are three cDNAs which do not have any exons irrelevantto the presumed translation region, and consist of a single exon(#10-#12, SEA ID NOS: 83-85).

1. A method for restoring rice fertility comprising introducing anucleic acid into rice, wherein the nucleic acid encodes the amino acidsequence of SEQ ID NO. 75, or an amino acid sequence which is identicalto at least 95% of the amino acid sequence of SEQ ID NO.
 75. 2. Themethod of claim 1, comprising introducing a nucleic acid into rice,wherein the nucleic acid encodes the amino acid sequence of SEQ ID NO.75.
 3. The method of claim 1 or 2, wherein the nucleic acid encoding theamino acid sequence of SEQ ID NO. 75, or an amino acid sequence which isidentical to at least 95% of the amino acid sequence of SEQ ID NO. 75 isselected from nucleic acids of the following a)-p): a) a nucleic acidcomprising the bases 215-2587 of SEQ ID NO:69; b) a nucleic acidcomprising the bases 213-2585 of SEQ ID NO:70; c) a nucleic acidcomprising the bases 218-2590 of SEQ ID NO:71; d) a nucleic acidcomprising the bases 208-2580 of SEQ ID NO:72; e) a nucleic acidcomprising the bases 149-2521 of SEQ ID NO:73; f) a nucleic acidcomprising the bases 225-2597 of SEQ ID NO:74; g) a nucleic acidcomprising the bases 43907-46279 of SEQ ID NO:27; h) a nucleic acidcomprising the bases 229-2601 of SEQ ID NO:80; i) a nucleic acidcomprising the bases 175-2547 of SEQ ID NO:81; j) a nucleic acidcomprising the bases 227-2599 of SEQ ID NO:82; k) a nucleic acidcomprising the bases 220-2592 of SEQ ID NO:83; l) a nucleic acidcomprising the bases 174-2546 of SEQ ID NO:84; m) a nucleic acidcomprising the bases 90-2462 of SEQ ID NO:85; n) a nucleic acid which isidentical to at least 95% of the nucleic acid of any of a)-m); o) anucleic acid which hybridizes to the nucleic acid of any of a)-m)underhybridization conditions of 0.1×SSC to 0.2×SSC at about 60-65° C.and/or washing conditions of 0.2×SSC, 0.1% SDS at about 65-68° C.; andp) a nucleic acid wherein one or a plurality of base(s) is deleted from,added to or substituted from the nucleic acid of any of a)-m).
 4. Themethod of claim 3, wherein the nucleic acid encoding the amino acidsequence of SEQ ID NO. 75, or an amino acid sequence which is identicalto at least 95% of the amino acid sequence of SEQ ID NO. 75, and whichmeets at least one of the following requirements 1)-12): 1) a basecorresponding to the base 1769 of SEQ ID NO. 69 is A; 2) a basecorresponding to the base 1767 of SEQ ID NO. 70 is A; 3) a basecorresponding to the base 1772 of SEQ ID NO. 71 is A; 4) a basecorresponding to the base 1762 of SEQ ID NO. 72 is A; 5) a basecorresponding to the base 1703 of SEQ ID NO. 73 is A; 6) a basecorresponding to the base 1779 of SEQ ID NO. 74 is A; 7) a basecorresponding to the base 1783 of SEQ ID NO. 80 is A; 8) a basecorresponding to the base 1729 of SEQ ID NO. 81 is A; 9) a basecorresponding to the base 1781 of SEQ ID NO. 82 is A; 10) a basecorresponding to the base 1774 of SEQ ID NO. 83 is A; 11) a basecorresponding to the base 1728 of SEQ ID NO. 84 is A; or 12) a basecorresponding to the base 1644 of SEQ ID NO. 85 is A.
 5. A method forrestoring rice fertility comprising introducing a nucleic acid intorice, wherein the nucleic acid comprises SEQ ID NO: 69 bases 215-2587,or a nucleic acid sequence comprising a sequence having at least 95%identity to the nucleic acid of SEQ ID NO. 69 bases 215-2587.